Bioreactors with substance injection capacity

ABSTRACT

A bioreactor with substance injection capability. In one embodiment, the bioreactor includes a first substrate having a first surface, an opposite second surface and edges. The bioreactor further includes a second substrate having a first surface and an opposite second surface, defining a cavity with a bottom surface, where the bottom surface is located therebetween the first surface and the second surface. The first surface of the first substrate is received by the second surface of the second substrate to cover the cavity so as to form a chamber for receiving cells and a liquid medium. A port is formed in the second substrate between the bottom surface and the first surface of the second substrate. As formed, the port is in fluid communication with the chamber to allow a stream of substance to be introduced into the chamber. The stream of substance is controlled so as to provide a gradient, or a concentration gradient of the substance, to the chamber. The stream of substance includes a substance affecting the growth of cells such as chemokine.

This application is being filed as a PCT International Patentapplication in the name of Vanderbilt University, a U.S. nationalcorporation, applicant for the designation of all countries except theUS, and John P. Wikswo and Franz J. Baudenbacher, both U.S. nationalsand residents, applicants for the designation of the US only, on 27 Aug.2003.

The present invention was made with Government support under Grant No.N66001-01-C-8064 awarded by the Defense Advanced Research ProjectsAdministration and the Office of Naval Research. The United StatesGovernment may have certain rights to this invention pursuant to thesegrants.

Some references, which may include patents, patent applications andvarious publications, are cited in a reference list and discussed in thedescription of this invention. The citation and/or discussion of suchreferences is provided merely to clarify the description of the presentinvention and is not an admission that any such reference is “prior art”to the invention described herein. In terms of notation, hereinafter,“[n]” represents the nth reference cited in the reference list. Forexample, [¹¹] represents the 11th reference cited in the reference list,namely, Hu, W. S. and Aunins, J. G., Large-Scale Mammalian Cell Culture,Curr. Opin. Biotechnol., 8, 148-153, 1997.

FIELD OF THE INVENTION

The present invention generally relates to an apparatus and methods forgrowing and maintaining a living system. More particularly, the presentinvention relates to an apparatus and methods that have a channelconfiguration allowing perfusate flow with diffusional exchange totissue cells but no cell migration. Additionally, the present inventionrelates to an apparatus and methods that have capacity for growing andmaintaining a living microorganism such as protozoa.

The present invention also relates to an apparatus and methods fordynamic analysis of a collection of cells such as a biofilm. Moreparticularly, the present invention relates to an apparatus and methodsfor measuring response of a biofilm to one or more dynamic streams ofsubstance such as chemical stressors at various depths of the biofilm.

Certain embodiments of the present invention comprise apparatus andmethods for growing and maintaining a living system such as a cell or acollection of cells and monitoring the status of such a living systemthat is metabolically active and responsive to environmental change,wherein each metabolic activity of the cell may be characterized by acharacteristic time. More particularly, the apparatus and methodscomprise bioreactors with multiple chambers and methods of using thesame.

Certain other embodiments of the present invention comprise apparatusand methods for growing and maintaining a living system such as a cellor a collection of cells and monitoring the status of such a livingsystem that is metabolically active and responsive to environmentalchange, wherein each metabolic activity of the cell may be characterizedby a characteristic time. More particularly, the apparatus and methodscomprise bioreactors with an array of chambers with a common feed lineand methods of using the same.

Certain additional embodiments of the present invention compriseapparatus and methods for growing and maintaining a living system suchas a cell or a collection of cells and monitoring the status of such aliving system that is metabolically active and responsive toenvironmental change, wherein each metabolic activity of the cell may becharacterized by a characteristic time. More particularly, the apparatusand methods comprise capillary perfused bioreactors and methods of usingthe same.

Certain further embodiments of the present invention comprise apparatusand methods for growing and maintaining a living system such as a cellor a collection of cells and monitoring the status of such a livingsystem that is metabolically active and responsive to environmentalchange, wherein each metabolic activity of the cell may be characterizedby a characteristic time. More particularly, the apparatus and methodscomprise bioreactors with substance injection capability and methods ofusing the same.

BACKGROUND OF THE INVENTION

Bioreactor is a device that can be used for culturing living cells. Moreparticularly, bioreactors are vessels that provide a proper physical andchemical environment as well as fast transport of substrates andproducts to allow cellular biological reactions to occur, ideallyrapidly and efficiently. The simplest bioreactor is a culture dish: Inconventional cell culture using well-plates, culture-dishes, and flasks,the volume of the culture medium is typically 200 to 1000 times thevolume of the cells. This ratio, when used in combination with bufferingof the culture media, allows the cells to grow for at least 24 hourswithout media change. However, another consequence of this ratio is acorresponding dilution of whatever extracellular factors are produced bythe cells and might otherwise provide paracrine cell-to-cellcommunication, which is possible in tissue because the extracellularvolume might be only 10% of intracellular volume.

Much of the development of bioreactors was directed towards either thefunctional tissues, or the generation of biochemicals andpharmaceuticals. For example, over the last 20 years studies on thegeneration of skin, pancreas, cartilage, liver, cornea and bladder havetaken particular importance¹. In the United States alone, there are morethan 80,000 individuals waiting for an organ transplant, and hence theneed to develop improved bioreactor technology is self-evident. There isalso a growing recognition that progress in understanding cell motilityand chemotactic signaling, as well as other complex cellular processes,is often constrained by the laboratory techniques available forobserving and intervening at various points in the processes. Many ofthese processes can be examined best in a properly instrumentedbioreactor.

There is a wide variety in bioreactors, including stirred vessels,bubble column, packed beds², air-lift reactors, and membrane reactors³that include plates, rotating plates, spiral-wound and hollow fibres.Hollow-fiber reactors are of special importance since (depending oftheir structure) they may allow as much as 30,000 m² of membrane areaper m³ module volume⁴⁻⁶. However, given that mammalian cells are verysensitive to shear forces⁷⁻⁹ (which originate mainly from agitation andaeration), it is important to reduce the forces as much as possible inthe reactor where the cells will be grown^(9,10). Membranes have beenused in bioreactors to increase survival of cells. For instance, it hasbeen known that liquid-gas interface created in some models of reactorsis particularly damaging for mammalian cells. That potentially lethalinterface can be eliminated by the use of a hydrophobic membrane⁹.

Bioreactors may be also classified by means of their mode of operation:batch, fed-batch and continuous cultivation (also called perfusedcultivation). In the first or batch mode, no substrate is added, normedium removed; in the case of the fed-batch mode there is a continuousfeeding, but nothing is removed until the reactions are terminated andthe reactor emptied. While these systems imply a low effort for processcontrol, the productivity is low compared to that in perfused systems,the third mode, where a permanent inflow of substrate and outflow ofmedium takes place. Besides the high productivity, there is a bettercell physiology control in this kind of reactors¹¹ and in the case ofmammalian cell culture, it has been shown to provide significantadvantage over static methods^(12,13).

One of the limitations when developing large three-dimensional tissuesis the lack of a proper vascular supply for nutrient and metabolitetransport. A number of studies have analyzed the artificial vascularnetworks¹⁴⁻¹⁸, and there have been a number of attempts to constructfunctional microfabricated scaffolds^(,16,19-21). The techniques bywhich these networks have been produced include plasma etching,photolithography, soft lithography, microcontact printing, microfluidicpatterning using microchannels, laminar flow patterning and stencilpatterning²²⁻²⁵. In the case of plasma etching technologies we canconsider the high aspect ratio micromachining (HARMS) as a very powerfultool since it allows to etch channels of virtually unlimited depthwithout increasing the width already achieved by lithography²². It isalso possible to construct three dimensional microchannel systems inPDMS with complex topologies and geometries's.

Additionally, one needs to realize that the growth ofclinically-implantable tissue may require the ultimate biodegradationand the mechanical properties of the tissue scaffold¹⁶. These propertiesare directly related to the crystallinity, molecular weight, glasstransition temperature and monomer hydrophobicity of the materialschosen to fabricate the tissue¹⁹. Naturally derived materials such ascollagen have been employed²⁶, as well as synthetic and semi syntheticones. Polyglycolic acid (PGA) possesses high porosity and it makes easythe fabrication of devices, therefore, PGA fibre meshes have beenconsidered to transplant cells. However, they cannot resist significantcompressional forces. An alternative to solve this problem is to usepolymers of lactic and glycolic acid whose ratios can be adjusted tocontrol the crystallinity of the material and hence the degradation rateand mechanical properties. Fibre-based tubes have been fabricated fromthese polymers²⁷.

It is important to compare the vascular nature of living tissue with thecapabilities provided by existing microfabricated cell-perfusionbioreactor systems. In tissue, arteries divide into progressivelysmaller vessels, eventually reaching arterioles and then capillaries.The arterioles are important because they contain the precapillarysphincters, which allow control of the perfusion of individual capillarybeds, but also provide the majority of the peripheral resistance andhence the pressure drop associated with the arterial supply. As aresult, the pressure difference across the capillary endotheliummembrane is kept sufficiently low to allow diffusional transport ofnutrients and metabolites across the membrane, as well as the trafficingof immune cells required for tissue maintenance and infection control.Were the pressures in the capillaries as high as those in thearterioles, the capillary wall thickness would be too great to allowthese critical transport phenomena. The venous return system is in manyways a mirror of the arterial system, albeit at lower pressures. Anotherfeature of the living vascular system is that the branching processdescribed above allows all cells to be within 50 to 200 microns of acapillary, depending upon the specific tissue. As a result, the arterialsupply and venous return systems are intercalated in such a manner thatevery capillary that perfuses a large group of cells is connected to thelarger supply and return systems with a self-similarity that ensuresuniform perfusion and transcapillary pressures. It is this intercalationprocess that is so difficult to replicate with microfabrication. Forexample, Borenstein et al.,²² describe a process to build atwo-dimensional vascular system that could create a multi-scaleperfusion system for supporting endothelial cells, but there is noprovision to selectively limit diffusive transport across the smallestcapillaries to perfuse cells lying outside of the perfusion network.More importantly, the networks they show have a large region of thedevice that is covered with the larger vessels, and the region of thebioreactor that is limited to capillary vessels is in fact quite small.

Thus, there is a need for microfabricated migration bioreactors thatmimic in vitro the microenvironments of normal tissue was well as thatof tumors, infected tissue, and wounded tissue, while providingindependent control of chemokine and growth factor gradients, shearforces, cellular perfusion, and the permeability of physical barriers tocellular migration, thereby allowing detailed optical andelectrochemical observation of normal, immune, and cancerous cellsduring cell migration, intravasation, extravasation, and angiogenesis.Angiogenesis, tumor metastasis, and leukocyte infiltration into tissueare complex processes that are regulated not only by cellular responsesto a single chemokine, but also by external factors, such as multiplecompeting chemokine and growth factor signals, autocrine feedback loops,cell-cell interactions, and mechanical forces such as vessel shearstress. Current approaches for assessing migration across cellularbarriers include Boyden and transwell chambers that provide anintegrated fluorescence assay of migration across filters to allowquantitation of migration²⁸⁻³⁴, parallel plate flow chambers³⁵⁻³⁸, inwhich adhesion and rolling on endothelial cells in shear stress can beassessed^(35,39-44), and in vivo intravital microscopy in whichmigration of cells in living animals is visualized⁴⁵⁻⁴⁸. Each of theseapproaches has limitations, including the inability to have sustainedand controlled chemotactic gradients (all systems), the inability tovisualize migration in real time or with physiologic shear stress(Boyden and transwell chambers), the inability to observe extravasationor angiogenesis into an underlying, deep cellular matrix (parallel plateflow chambers) and the inability to control all aspects of theexperiments, e.g., having defined cell populations and controlledmicrofluidics for independent control of shear and tissue perfusion (allsystems, especially intravital microscopy). The development of amotility/metastasis model system with independent control of endothelialshear stress, chemokine gradients, tissue perfusion, and the ability toadd different cell types through different ports, combined with state-ofthe art imaging techniques and sensor capabilities would represent ahuge advance over currently available systems.

Indeed, the need for such capabilities is quite urgent. Angiogenesis isa dynamic process, influenced by the cellular microenvironment andintricately linked to metastasis^(49,50). It has been demonstrated thatboth VEGF and angiopoietin/tyrosine kinase (Ang/Tie2) function arerequired for tumor angiogenesis⁵¹⁻⁵³. However, how signals from thosetwo receptor systems are integrated to mediate angiogenesis has not beendetermined, in part due to the lack of good model systems. The next stepwould be to study the coordination and integration of VEGF and Angsignaling in endothelial cell migration, vascular sprouting andmaturation, and tumor transendothelium migration. As with angiogenesis,multiple environmental inputs affect tumor metastasis and leukocyteinfiltration. Activation of one chemokine receptor in tumor cellsaffects the induction of other ligands and receptors in tumor cells aswell as endothelial cells and leukocytes, but the mechanism is poorlyunderstood⁵⁴. There is a need for an understanding of how alteration ofchemokine receptor internalization and/or changes in receptorassociation with adaptor molecules such as AP-2 or beta-arresting affectchemokine receptor activity as tumor cells move through a complexmatrix. How external factors such as cell-cell adhesion, cell-matrixinteractions, and vessel shear stress affect cytoskeletal reorganizationduring migration through tissues is also poorly understood. Cortactinoverexpression increases the metastasis of breast cancer cells tobone⁵⁵, however the mechanism remains unclear. Likewise, lack of WASpprotein in humans leads to an X-linked immune disorder that may resultfrom signaling, proliferation or chemotaxis defects⁵⁶. There is a needto study the role of cortactin and WASp proteins in chemotaxis of breastcancer and HL60 cells in a complex multicell environment involvingcontrollable shear, cell-cell interactions, and chemokine gradients. Asa final example, matrix metalloproteinases (MMPs) are extracellularlyexpressed enzymes found in many types of cancer and are thought to beimportant in tumor development, growth, invasion and metastasis. It hasrecently been discovered that skin tumors that develop in mice deficientfor MMP-3 (MMP-3 null mice) progress and grow much faster than skintumors from normal, wild-type mice. This difference is associated with areduced number of immune cells in the tumor and surrounding tissue inthe MMP-3 null mice. The logical progression of this research is todetermine how loss of an MMP affects the ability of immune cells, namelymonocytes and neutrophils, to infiltrate from the peripheral bloodcirculation to the tumor site. The ability to control the experimentalenvironment, including multiple defined cell populations, is critical toelucidate the relative importance of tumor-host interactions in MMP-3induced cellular chemotaxis.

Despite the progress made over the years, however, currently availablebioreactors cannot provide a more physiologic environment that wouldinclude a three-dimensional in vitro region with multiple cell types,stimuli, and measurement capabilities and allows study of molecularaspects of the chemotactic response. Thus, bioreactors that mimic invitro the microenvironments of tumors and tissue while providingindependent control of chemokine and growth factor gradients, shearforces, cellular perfusion, and the permeability of physical barriers tocellular migration, thereby allowing detailed optical andelectrochemical observation of normal and cancerous cells during cellmigration, intravasation, extravasation, and angiogenesis need to bedeveloped.

Therefore, a heretofore unaddressed need exists in the art to addressthe aforementioned deficiencies and inadequacies.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a bioreactor withsubstance injection capability. In one embodiment, the bioreactorincludes a first substrate having a first surface, an opposite secondsurface and edges. The bioreactor further includes a second substratehaving a first surface and an opposite second surface, defining a cavitywith a bottom surface, where the bottom surface is located therebetweenthe first surface and the second surface. The first surface of the firstsubstrate is received by the second surface of the second substrate tocover the cavity so as to form a chamber for receiving cells and aliquid medium. The second substrate can be fabricated from glass, Mylar,PDMS, silicon, a polymer, a semiconductor, or any combination of them.

A port is formed in the second substrate between the bottom surface andthe first surface of the second substrate with a first opening and anopposite, second opening. As formed, the port is in fluid communicationwith the chamber through the first opening to allow a stream ofsubstance to be introduced into the chamber through the portsubstantially along a first direction. The stream of substance iscontrolled so as to provide a gradient, or a concentration gradient ofthe substance, to the chamber at least around the first opening. Thestream of substance includes a substance affecting the growth of cellssuch as chemokine.

The second substrate further defines a third opening and an oppositefourth opening adapted for allowing a flow of liquid to be introducedinto the chamber through the third opening and away from the chamberthrough the fourth opening substantially along a second direction. Thesecond direction is substantially perpendicular to the first direction.

The bioreactor further includes a biocompatible coating layer applied tothe bottom surface of the second substrate. The biocompatible coatinglayer includes a material that may inhibit cell adhesion to thebiocompatible coating layer, enhance cell adhesion to the biocompatiblecoating layer, or function as a fluorescent marker or indicator of thestate of cells.

In forming the bioreactor, the first surface of the first substrate andthe second surface of the second substrate is spaced such that when alayer of cells grows on the biocompatible coating layer, a flow ofliquid can flow in the chamber between the first surface of the firstsubstrate and the layer of cells along the second direction. The flow ofliquid is controlled so as to provide a known shear force to the layerof cells. The flow of liquid may be further controlled so as to provideperfusion and maintenance to the layer of cells. In other words, thisflow can perfuse all cells in the chamber, and can be intermittent onlyas allowed by cell maintenance. The cells can be any type of livingcells, including, but not limited to, bacteria, protozoa, or both,normal cells, tumor cells, or any combination of them. Cells can beintroduced into a chamber individually, in a collection of cells, or inthe form of biofilm.

Moreover, the first surface of the first substrate and the secondsurface of the second substrate are spaced to further allow at least onecell to migrate above the layer of cells. The at least one cell tomigrate can be a cell having a type that is the same or different fromthe type of the layer of cells.

In an alternative embodiment of the present invention, the bioreactorfurther includes a layer of porous material that is positioned on thebottom surface of the second substrate. A biocompatible coating layercan be applied to the layer of porous material such that the layer ofporous material is between the biocompatible coating layer and thebottom surface of the second substrate. The biocompatible coating layerincludes a material that may inhibit cell adhesion to the biocompatiblecoating layer, enhance cell adhesion to the biocompatible coating layer,or function as a fluorescent marker or indicator of the state of cells.

In this embodiment, the first surface of the first substrate and thesecond surface of the second substrate are spaced such that when a layerof cells grows on the biocompatible coating layer, a flow of liquid canflow in the chamber between the first surface of the first substrate andthe layer of cells. The flow of liquid can also be controlled so as toprovide a known shear force to the layer of cells. As such formed, thechamber is divided by the biocompatible coating layer into two regions:an upper region for flow, and a lower region for cell extravasationand/or other cell activities.

The layer of porous material can include collagen, an extracellularmatrix, at least one cell culture scaffold supportive to the layer ofcells, or any combination of them. The layer of porous material mayallow at least one cell to extravasate below the layer of cells.

The first substrate is at least partially optically transparent. Abiocompatible coating layer may be applied to the first surface of thefirst substrate, where the biocompatible coating layer includes amaterial that may inhibit cell adhesion to the biocompatible coatinglayer, enhance cell adhesion to the biocompatible coating layer, orfunction as a fluorescent marker or indicator of the state of cells.

The first substrate and the second substrate are substantially parallelto each other and a plurality of posts are positioned between the firstsurface of the first substrate and the second surface of the secondsubstrate to substantially maintain a predetermined separation betweenthe first surface of the first substrate and the second surface of thesecond substrate to allow optical detecting of dynamic activities ofcells in the chamber. The dynamic activities of cells in the chamber aredetectable through optical detecting means such as high-resolutionoptical microscope or a fluorescence-imaging device or both.

The predetermined separation between the first surface of the firstsubstrate and the second surface of the second substrate should bemaintained with sufficient accuracy for accurate optical measurements.To this end, the plurality of posts are positioned in at least two rows,and wherein each row of posts has at least two posts spaced from eachother to form a stable support structure.

In an alternative embodiment, a bioreactor with a chamber is providedwith perfusion means in fluid communication with the chamber to allowdiffusional exchange of nutrients and metabolic byproducts with thechamber.

The perfusion means includes a nanofilter with a plurality of pores influid communication with the chamber, wherein the pores are sized toallow diffusional exchange of nutrients and metabolic byproducts withthe chamber and not to allow cells to migrate across the nanofilter. Thepores may be further sized to allow cells to perfuse through only bybi-directional diffusion through the nanofilter in a manner such thatsubstantially no shear is generated by the perfusion of cells. In oneembodiment, the pores of the nanofilter are sized to have a dimensionsmaller than 400 nanometers cross-sectionally.

The perfusion means further includes a perfusion supply network in fluidcommunication with the nanofilter through the pores. In one embodiment,the perfusion supply network includes a plurality of perfusion channels,each being in fluid communication with the nanofilter to allowbidirectional, diffusional exchange of nutrients and metabolicbyproducts with the nanofilter and being dimensioned to minimizepressure drops along each perfusion channel and to allow passivediffusional exchange of nutrients and metabolic byproducts along eachperfusion channel.

The perfusion supply network further includes a plurality ofintermediate supply channels, each being in fluid communication with aplurality of corresponding perfusion channels so as to provide perfusateto the plurality of corresponding perfusion channels. Moreover, theperfusion supply network has a plurality of intermediate returnchannels, each being in fluid communication with a plurality ofcorresponding perfusion channels so as to collect perfusate from theplurality of corresponding perfusion channels.

Additionally, the perfusion supply network further includes a pluralityof main supply channels, each being in fluid communication with aplurality of corresponding intermediate supply channels so as to provideperfusate to the plurality of corresponding intermediate supplychannels, and a plurality of main return channels, each being in fluidcommunication with a plurality of corresponding intermediate returnchannels so as to collect perfusate from the plurality of correspondingintermediate return channels.

In another aspect, the present invention relates to yet anotherbioreactor for cultivating living cells in a liquid medium. In oneembodiment, the bioreactor includes a first substrate having a firstsurface, an opposite second surface and edges. The bioreactor furtherincludes a second substrate having a first surface and an oppositesecond surface, defining a cavity with a bottom surface, where thebottom surface is located therebetween the first surface and the secondsurface. The first surface of the first substrate is received by thesecond surface of the second substrate to cover the cavity so as to forma chamber for receiving cells and a liquid medium. The second substratecan be fabricated from glass, Mylar, PDMS, silicon, a polymer, asemiconductor, or any combination of them.

The bioreactor further includes a filter dividing the chamber into afirst subchamber and a second subchamber, wherein the filter has aporosity to allow the first subchamber and the second subchamber influid communication. Additionally, a port is formed in the secondsubstrate between the bottom surface and the first surface of the secondsubstrate with a first opening and an opposite, second opening. Asformed, the port is in fluid communication with the second subchamberthrough the first opening to allow a stream of substance to beintroduced into the chamber through the port substantially along a firstdirection. The stream of substance is controlled so as to provide agradient, or a concentration gradient of the substance, to the chamberat least around the first opening. The stream of substance includes asubstance affecting the growth of cells such as chemokine.

The second substrate further defines a third opening and an oppositefourth opening adapted for allowing a flow of liquid to be introducedinto at least one of the first subchamber and the second subchamberthrough the third opening and away from at least one of the firstsubchamber and the second subchamber through the fourth openingsubstantially along a second direction. The second direction issubstantially perpendicular to the first direction. Same or differentflows of liquid can be introduced to one or both of the first subchamberand the second subchamber, jointly or independently.

The filter has a first surface that partially defines the firstsubchamber with the first surface of the first substrate, and anopposite second surface that partially defines the second subchamberwith the second surface of the second substrate. The filter includes aperfusion membrane with a plurality of pores to allow the filter to bein fluid communication with one or both of the first subchamber and thesecond subchamber. The pores of the filter are sized to allowdiffusional exchange of nutrients and metabolic byproducts with one orboth of the first subchamber and the second subchamber but not to allowcells to migrate across the filter. The pores are further sized to allowcells to perfuse through the filter only by bi-directional diffusion ina manner such that substantially no shear is generated by the perfusionof cells. In one embodiment, the pores of the filter are sized to have adimension smaller than 400 nanometers cross-sectionally. In a morepreferred embodiment, the pores of the filter are sized to have adimension about 10 to 100 nanometers cross-sectionally.

The bioreactor further includes a plurality of posts that arestrategically positioned between the first surface of the firstsubstrate and the first surface of the filter to substantially maintaina predetermined separation between the first surface of the firstsubstrate and the first surface of the filter to allow optical detectingof dynamic activities of cells in the first subchamber. Additionally,the bioreactor includes a plurality of posts that are strategicallypositioned between the second surface of the second substrate and thesecond surface of the filter to substantially maintain a predeterminedseparation between the second surface of the second substrate and thesecond surface of the filter to allow optical detecting of dynamicactivities of cells in the second subchamber.

The predetermined separation between the first surface of the firstsubstrate and the first surface of the filter and the predeterminedseparation between the second surface of the second substrate and thesecond surface of the filter should be maintained with sufficientaccuracy for accurate optical measurements, respectively. To this end,the plurality of posts and are positioned in at least two rows,respectively, and where each row of posts has at least two posts spacedfrom each other to form a stable support structure. Posts may bepositioned away from each other.

As such formed, when a first flow of liquid is introduced into the firstsubchamber, the first flow of liquid can be controlled so as to providea known shear force to a first layer of cells growing in the firstsubchamber on the first surface side of the filter and an environmentthat simulates a vascular space in the first subchamber. Jointly orindependently, a second flow of liquid can also be introduced into thesecond subchamber, and the second flow of liquid can be controlled so asto provide an environment that simulates a tissue space in the secondsubchamber. The fact that first flow of liquid and the second flow ofliquid can be controlled independently from each other means, amongother things, they can have different contents, different flowvelocities, and/or different timing of flow.

Moreover, as such formed, the bioreactor allows growing and culture ofmultiple layers (or populations) of cells therein. In addition to thefirst layer of cells growing in the first subchamber, a second layer ofcells is capable of growing in the second subchamber on the secondsurface side of the filter. The first layer of cells growing in thefirst subchamber and the second layer of cells growing in the secondsubchamber can be the same or different.

In yet another embodiment, an extension port member defining a channeltherein is provided. As formed, the extension port member is positionedcomplimentarily to the port such that the channel of the extension portmember is in fluid communication with the port and the first subchamberto allow the stream of substance to be directly introduced to the firstsubchamber.

In another embodiment, a bioreactor is provided with a first subchamberand a second subchamber, which are divided by a first filter, anextension port member and perfusion means in fluid communication with atleast one of the first subchamber and the second subchamber to allowdiffusional exchange of nutrients and metabolic byproducts with one orboth the first subchamber and the second subchamber.

The perfusion means includes a second filter (or nanofilter), with aplurality of pores in fluid communication with the second subchamber,wherein the pores are sized to allow diffusional exchange of nutrientsand metabolic byproducts with the second subchamber and not to allowcells to migrate across the second filter. The pores of the secondfilter, for example, can be sized to have a dimension smaller than 400nanometers cross-sectionally. The first filter and the second filter canbe the same or different.

The perfusion means further includes a perfusion supply network in fluidcommunication with the second filter through the pores. In oneembodiment, the perfusion supply network includes a plurality ofperfusion channels, each being in fluid communication with the secondfilter to allow bidirectional, diffusional exchange of nutrients andmetabolic byproducts with the second filter and being dimensioned tominimize pressure drops along each perfusion channel and to allowpassive diffusional exchange of nutrients and metabolic byproducts alongeach perfusion channel.

The perfusion supply network additionally includes a plurality ofintermediate supply channels, each being in fluid communication with aplurality of corresponding perfusion channels so as to provide perfusateto the plurality of corresponding perfusion channels. Moreover,perfusion supply network includes a plurality of intermediate returnchannels, each being in fluid communication with a plurality ofcorresponding perfusion channels so as to collect perfusate from theplurality of corresponding perfusion channels.

Furthermore, the perfusion supply network includes a plurality of mainsupply channels, each being in fluid communication with a plurality ofcorresponding intermediate supply channels so as to provide perfusate tothe plurality of corresponding intermediate supply channels, and aplurality of main return channels, each being in fluid communicationwith a plurality of corresponding intermediate return channels so as tocollect perfusate from the plurality of corresponding intermediatereturn channels.

This type of bioreactor with a microfabricated transwell chamber withchemokine injection capability can be utilized to supported a coculturewith filter perfusion to allow independent control of perfusion andshear in the chamber. The perfusion means maintains the viability ofcells in the lower chamber independent of the flow in the upper chamber.Stream of substances affecting growth of cells such as chemotacticagents can be injected through dedicated ports in the perfusion means.

Optionally, at least one insertion member defining a cavity therein isprovided. The insertion member has a length L and is positioned throughthe second substrate and into the tissue space such that the cavity ofthe insertion member is in fluid communication with the first subchamberor the vascular space.

Correspondingly, a plug having a first surface and an opposite secondsurface is provided. The plug is complimentary to a correspondinginsertion member such that when the plug is received into the cavity ofthe corresponding insertion member, the plug engages with the body ofthe corresponding insertion member to seal the cavity and a volume isformed between the first surface and the first filter to allow acollection of cells to be received therein. For example, a collection oftumor cells can be contained in the volume. Optionally, a cage adaptedfor separating the tumor cells from the first subchamber can beutilized.

Additionally, the plug further defines a port in fluid communicationwith the volume for injecting or withdrawing a stream of substanceaffecting the growth of the tumor cells such as chemokine. Moreover, aplurality of electrodes adapted for electrochemical measurements of thetumor cells can be utilized together with the plug to form a metabolicsensing head.

In yet another alternative embodiment, a bioreactor is provided with anextension port member defining a channel therein. The extension portmember is positioned such that the channel of the extension port memberis in fluid communication with the first subchamber to allow a stream ofsubstance to be introduced to the first subchamber or the vascularspace. For example, a gradient of chemokine can be introduced into thefirst subchamber or the vascular space. A similar structure can beutilized to provide a stream of substance such as a gradient ofchemokine to the tissue space.

These and other aspects of the present invention will become apparentfrom the following description of the preferred embodiment taken inconjunction with the following drawings, although variations andmodifications therein may be affected without departing from the spiritand scope of the novel concepts of the disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A schematically shows a top view of a bioreactor with a barrieraccording to one embodiment of the present invention.

FIG. 1B shows a perspective view of a bioreactor with a barrieraccording to another embodiment of the present invention.

FIG. 2A schematically shows a bioreactor with a common feed lineaccording to one embodiments of the present invention.

FIG. 2B shows a cross-sectional partial view of a bioreactor as shown inFIG. 1A.

FIG. 3 shows a cross-sectional partial view of a bioreactor with acommon feed line according to another embodiment of the presentinvention.

FIGS. 4A-B shows a bioreactor with a common feed line according to yetanother embodiment of the present invention: 4A, a cross-sectionalpartial view, 4B, a cross-sectional side view along line A-A in FIG. 4A.

FIG. 5 shows a cross-sectional partial view of a bioreactor with acommon feed line according to a further embodiment of the presentinvention.

FIG. 6 shows a cross-sectional partial view of a bioreactor with acommon feed line according to a yet another single-array embodiment ofthe present invention.

FIG. 7 schematically shows a top view of a bioreactor with two barriersaccording to one embodiment of the present invention.

FIGS. 8A-D schematically show a top cross-sectional view of a bioreactorwith a multi-chamber according to one embodiment of the presentinvention.

FIG. 9A shows a perspective view of a layered perfusion system accordingto one embodiment of the present invention: the insert, a cut-off topplan view.

FIG. 9B shows a top view of different layers of a layered perfusionsystem as shown in FIG. 9A.

FIG. 9C shows an exploded view of a layered perfusion system as shown inFIG. 9A.

FIGS. 9D1-3 show a layered perfusion system as shown in FIG. 9A: 9D1, atop cross-sectional view, 9D2, a side cross-sectional view along lineB-B, 9D3, a side cross-sectional view along line A-A.

FIGS. 9E1-2 shows an electon micrograph of a PS-b-PMMA film deposited onsilicon: 9E1, at one magnification rate, 9E2, at another magnificationrate.

FIG. 9F shows an atomic force microscope image of a PS film template ona silicon wafer formed by spin casting a PS-b-PMMA film and removing thePMMA.

FIGS. 9GA-G5 schematically show a fabrication process of the layeredperfusion system as shown in FIG. 9A: 9G1, a side cross-sectionalpartial view of a silicon substrate, 9G2, step 1, etching channels 904 cinto the silicon wafer 953, 9G3, step 2, patterning the layer 951, 9G4,step 3, etching pores through the layer 952, 9G5, three views of thecompleted silicon substrate 950.

FIGS. 10A1-2 schematically show a bioreactor with multiple trapsaccording to one embodiment of the present invention: 10A1, aperspective view; and 10A2, a perspective sectional view.

FIG. 10B schematically shows a perspective view of a bioreactor withmultiple traps according to another embodiment of the present invention.

FIG. 10C schematically shows a top cross-sectional view of a bioreactorwith a confined region according to one embodiment of the presentinvention.

FIG. 10D schematically shows a top cross-sectional view of a bioreactorwith a confined region according to another embodiment of the presentinvention.

FIG. 10E schematically shows a top cross-sectional view of a bioreactorwith a confined region according to yet another embodiment of thepresent invention.

FIG. 10F schematically shows a top cross-sectional view of a bioreactorwith a confined region according to a further embodiment of the presentinvention.

FIG. 10G schematically shows a top cross-sectional view of a bioreactorwith multiple traps according to one embodiment of the presentinvention.

FIG. 10H schematically shows a top cross-sectional view of a bioreactorwith multiple traps according to another embodiment of the presentinvention

FIG. 10I schematically shows a top cross-sectional view of a bioreactorwith multiple traps according to yet another embodiment of the presentinvention.

FIGS. 11A1-3 schematically show a bioreactor according to one embodimentof the present invention: 11A1, a top cross-sectional view; 11A2, atransverse cross-sectional view; and 11A3, a lateral cross-sectionalview.

FIG. 11B schematically shows a top cross-sectional view of a bioreactoraccording to another embodiment of the present invention.

FIG. 11C schematically shows a top cross-sectional view of a bioreactoraccording to yet another embodiment of the present invention.

FIG. 11D schematically shows a top cross-sectional view of a bioreactoraccording to a further embodiment of the present invention.

FIGS. 12A1-3 show a bioreactor with chemokine injection according to oneembodiment of the present invention: 12A1, a top cross-sectional view,12A2, a side cross-sectional view, 12A3, another side cross-sectionalview.

FIGS. 12B shows a side cross-sectional view of a bioreactor withchemokine injection according to another embodiment of the presentinvention.

FIGS. 12C shows a side cross-sectional view of a bioreactor withchemokine injection according to yet another embodiment of the presentinvention.

FIGS. 12D shows a side cross-sectional view of a bioreactor withchemokine injection according to a further embodiment of the presentinvention.

FIGS. 13A shows a side cross-sectional view of a bioreactor with atwo-chamber according to one embodiment of the present invention.

FIGS. 13B shows a side cross-sectional view of the bioreactor as shownin FIG. 13A.

FIGS. 13C shows a side cross-sectional view of a bioreactor with atwo-chamber according to another embodiment of the present invention.

FIGS. 13D shows a side cross-sectional view of a bioreactor with asingle chamber according to one embodiment of the present invention.

FIGS. 13E shows a side cross-sectional view of a bioreactor with asingle chamber according to another embodiment of the present invention.

FIGS. 13F shows a side cross-sectional view of a bioreactor with asingle chamber according to yet another embodiment of the presentinvention.

DETAILED DESCRIPTION OF THE INVENTION

Various embodiments of the invention are now described in detail.Referring to the drawings, like numbers indicate like parts throughoutthe views unless the context clearly dictates otherwise. As used in thedescription herein and throughout the claims that follow, the meaning of“a,” “an,” and “the” includes plural reference unless the contextclearly dictates otherwise. Also, as used in the description herein andthroughout the claims that follow, the meaning of “in” includes “in” and“on” unless the context clearly dictates otherwise. Additionally, someterms used in this specification are more specifically defined below.

DEFINITIONS

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. For example, conventionaltechniques of molecular biology, microbiology and recombinant DNAtechniques may be employed in accordance with the present invention.Such techniques and the meanings of terms associated therewith areexplained fully in the literature. See, for example, Sambrook, Fitsch &Maniatis. Molecular Cloning: A Laboratory Manual, Second Edition (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referredto herein as “Sambrook et al., 1989”); DNA Cloning: A PracticalApproach, Volumes I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins, eds. 1984); Animal Cell Culture (R. I. Freshney, ed.1986); Immobilized Cells and Enzymes (IRL Press, 1986); B. E. Perbal, APractical Guide to Molecular Cloning (1984); F. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994). See also, PCR Protocols: A Guide to Methods and Applications,Innis et al., eds., Academic Press, Inc., New York (1990); Saiki et al.,Science 1988, 239:487; and PCR Technology: Principles and Applicationsfor DNA Amplification, H. Erlich, Ed., Stockton Press.

Certain terms that are used to describe the invention are discussedbelow, or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the devices and methods of theinvention and how to make and use them. For convenience, certain termsare highlighted, for example using italics and/or quotation marks. Theuse of highlighting has no influence on the scope and meaning of a term;the scope and meaning of a term is the same, in the same context,whether or not it is highlighted. It will be appreciated that the samething can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification, including examples of any terms discussed herein, isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

As used herein, “about” or “approximately” shall generally mean within20 percent, preferably within 10 percent, and more preferably within 5percent of a given value or range. Numerical quantities given herein areapproximate, meaning that the term “about” or “approximately” can beinferred if not expressly stated.

The term “molecule” means any distinct or distinguishable structuralunit of matter comprising one or more atoms, and includes for examplepolypeptides and polynucleotides.

As used herein, “cell” means any cell or cells, as well as viruses orany other particles having a microscopic size, e.g. a size that issimilar to that of a biological cell, and includes any prokaryotic oreukaryotic cell, e.g., bacteria, fungi, plant and animal cells. Cellsare typically spherical, but can also be elongated, flattened,deformable and asymmetrical, i.e., non-spherical. The size or diameterof a cell typically ranges from about 0.1 to 120 microns, and typicallyis from about 1 to 50 microns. A cell may be living or dead. As usedherein, a cell is generally living unless otherwise indicated. As usedherein, a cell may be charged or uncharged. For example, charged beadsmay be used to facilitate flow or detection, or as a reporter.Biological cells, living or dead, may be charged for example by using asurfactant, such as SDS (sodium dodecyl sulfate). Cell or a plurality ofcells can also comprise cell lines. Example of cell lines include livercell, macrophage cell, neuroblastoma cell, endothelial cell, intestinecell, hybridoma, CHO, fibroblast cell lines, red blood cells,electrically excitable cells, e.g. Cardiac cell, myocytes (ATI cells),cells grown in co-culture, NG108-15 cells (a widely used neuroblastoma Xglioma hybrid cell line, ATCC# HB-12317), primary neurons, a primarycardiac myocyte isolated from either the ventricles or atria of ananimal neonate, an AT-1 atrial tumor cardiac cell, Liver cells are alsoknown as Hepatocytes, Secretory cell (depolarize and it secretes things)pancreatic beta cells secrete insulin, HELA cells (Helen Lane), HEK293Human Epithial Kidney c, Erythrocytes (primary red blood cells),Lymphocytes and the like. Each cell line may include one or more cells,same or different. For examples, the liver cell comprises at least oneof Human hepatocellular carcinoma (“HEPG2”) cell, CCL-13 cell, and H4IIEcell, the macrophage cells comprises at least one of peripheral bloodmononuclear cells (“PBMC”), and skin fibroblast cells, the neuroblastomacell comprises a U937 cell, the endothelial cell comprises a humanumbilical vein-endothelial cell (“Huv-ec-c”), and the intestine cellcomprises a CCL-6 cell.

“Culture” means a growth of living cells in a controlled artificialenvironment. It may be a culture of microorganisms, such as a bacterialculture, or one of animal or plant cells, such as a tissue culture. Thebioreactors according to the invention can do both and more. Culturesrequire appropriate sources of food and energy, provided by the culturemedium, and a suitable physical environment. Tissue cultures canthemselves become a culture medium for viruses, which grow only withlive cells. Cultures of only one kind of cells are known as purecultures, as distinguished from mixed or contaminated cultures.

“Tissue” means an aggregation of cells more or less similarmorphologically and functionally. The animal body is composed of fourprimary tissues, namely, epithelium, connective tissue (including bone,cartilage, and blood), muscle, and nervous tissue. The process ofdifferentiation and maturation of tissues is called histogenesis.

A “sensor” is broadly defined as any device that can measure ameasurable quantity. For examples, a sensor can be a thermal detector,an electrical detector, a chemical detector, an optical detector, an iondetector, a biological detector, a radioisotope detector, anelectrochemical detector, a radiation detector, an acoustic detector, amagnetic detector, a capacitive detector, a pressure detector, anultrasonic detector, an infrared detector, a microwave motion detector,a radar detector, an electric eye, an image sensor, any combination ofthem and the like. A variety of sensors can be chosen to practice thepresent invention.

The term “analyte” means a material that can be consumed or produced bya cell. Examples of analyte of interest include pH, K, oxygen, lactate,glucose, ascorbate, serotonin, dopamine, ammonina, glutamate, purine,calcium, sodium, potassium, NADH, protons, insulin, NO (nitric oxide)and the like.

The term “flow” means any movement of fluid such as a liquid or solidthrough a device or in a method of the invention, and encompasseswithout limitation any fluid stream, and any material moving with,within or against the stream, whether or not the material is carried bythe stream. For example, the movement of molecules or cells through adevice or in a method of the invention, e.g. through channels of asubstrate on microfluidic chip of the invention, comprises a flow. Thisis so, according to the invention, whether or not the molecules or cellsare carried by a stream of fluid also comprising a flow, or whether themolecules or cells are caused to move by some other direct or indirectforce or motivation, and whether or not the nature of any motivatingforce is known or understood. The application of any force may be usedto provide a flow, including without limitation, pressure, capillaryaction, electroosmosis, electrophoresis, dielectrophoresis, opticaltweezers, and combinations thereof, without regard for any particulartheory or mechanism of action, so long as molecules or cells aredirected for detection, measurement or sorting according to theinvention.

A “liquid or medium” is a fluid that may contain one or more substancesthat affecting growth of cells, one or more analytes, or any combinationof them. A medium can be provided with one or more analytes to beconsumed by one or more cells. A medium can have one or more analytesgenerated by one or more cells. A medium can also have at the same timeone or more analytes to be consumed by one or more cells and one or moreanalytes generated by one or more cells. A medium may consist of naturalmaterials, such as enzymatic digests, extracts of yeast or beef, milk,potato slices, or chick embryos. Artificial media are prepared by mixingvarious ingredients according to particular formulas. A complex mediumcontains at least one crude ingredient derived from a natural material,hence of unknown chemical composition. A chemically defined or syntheticmedium is one in which the chemical structure and amount of eachcomponent are known.

An “inlet region” is an area of a bioreactor that receives molecules orcells or liquid. The inlet region may contain an inlet port and channel,a well or reservoir, an opening, and other features which facilitate theentry of molecules or cells into the device. A bioreactor may containmore than one inlet region if desired. The inlet region is in fluidcommunication with the channel and is upstream therefrom.

An “outlet region” is an area of a bioreactor that collects or dispensesmolecules or cells or liquid. An outlet region is downstream from adiscrimination region, and may contain outlet channels or ports. Abioreactor may contain more than one outlet region if desired.

An “analysis unit” is a microfabricated substrate, e.g., amicrofabricated chip, having at least one inlet region, at least onechannel and chamber, at least one detection region and at least oneoutlet region. A device of the invention may comprise a plurality ofanalysis units.

A “channel” is a pathway of a bioreactor of the invention that permitsthe flow of molecules or cells to pass a detection region for detection(identification), or measurement. The detection and discriminationregions can be placed or fabricated into the channel. The channel istypically in fluid communication with an inlet port or inlet region,which permits the flow of molecules or cells or liquids into thechannel. The channel is also typically in fluid communication with anoutlet region or outlet port, which permits the flow of molecules orcells or liquid out of the channel. The channel can also be used as achamber to grown cells, and vice versa.

A “detection region” or “sensing volume” or “chamber” is a locationwithin the bioreactor, typically in or coincident with the channel (or aportion thereof) and/or in or coincident with a detection loop, wheremolecules or cells to be grown, identified, characterized, hybridized,measured, analyzed or maintained (etc.), are examined on the basis of apredetermined characteristic. In one embodiment, molecules or cells areexamined one at a time. In other embodiments, molecules, cells orsamples are examined together, for example in groups, in arrays, inrapid, simultaneous or contemporaneous serial or parallel arrangements,or by affinity chromatography.

“Reaction time” is the time that a system of interest requires torespond to a change. For example, the reaction time of a cell is thetime required for at least one of the physiological processes of a cellto adapt or respond to a change in its environment. Each type of cellhas its own characteristic reaction time with respect to a particularchange in its environment. The reaction time of a sensor is the timerequired for the sensor to respond to a change in the quantity that itis sensing. For example, the reaction time of an electrochemical sensoris set by the size of the sensor and the thickness and nature ofprotective coatings on the activated surfaces of the sensor. Thereaction time of a microfluidic system is determined by, among otherthings, the reaction time of the cell to changes in the environment, thetime required for chemical species to diffuse throughout the sensingvolume, the reaction time of the sensor(s) and the diffusion time of theanalyte being controlled by the actuators.

“Bacteria” are extremely small—usually 0.3-2.0 micrometers indiameter—and relatively simple microorganisms possessing the prokaryotictype of cell construction. Each bacterial cell arises either by divisionof a preexisting cell with similar characteristics, or throughcombination of elements from two such cells in a sexual process.

“Protozoa” means a group of eukaryotic microorganisms traditionallyclassified in the animal kingdom. Although the name signifies primitiveanimals, some Protozoa (phytoflagellates and slime molds) show enoughplantlike characteristics to justify claims that they are plants.Protozoa range in size from 1 to 10⁶ micrometers. Colonies are known inflagellates, ciliates, and Sarcodina. Although marked differentiation ofthe reproductive and somatic zooids characterizes certain colonies, suchas Volvox, Protozoa have not developed tissues and organs.

Several embodiments are now described with reference to the FIGS. 1-2,in which like numbers indicate like parts throughout the FIGS. 1-2.

OVERVIEW OF THE INVENTION

The inventors of the present invention overcome the disadvantages of theprior art and develop new bioreactors that have, among other new andinventive features, the capability of providing controlled chemokinegradients independent of the perfusion flow and allow extravasation of acellular matrix. Recent advances in the fabrication of nanofilters⁵⁷⁻⁶¹are used to create perfused-membrane bioreactors according to thepresent invention that allow the growth of mixed cultures of cells atnear-to-tissue densities in 1 mm×1 mm×100 micron volumes, in thepresence of controlled, stable chemokine or growth-factor gradientswithin the device, to mimic the in vivo tumor microenvironment.

One advantage of the present invention is that custom devices can beconstructed such that the isolated perfusion and cell-delivery systemsallow independent control of shear stress and chemokine gradients duringthe course of an experiment. Moreover, the optical and electrochemicalmetabolic microsensors can be installed within these bioreactors toallow simultaneous quantification of the local metabolic and chemicalenvironment (lactate, pH, O₂, etc.) in selected regions within thereactor, while cell migration or cell signaling events are imaged byfluorescence microscopy. Hence, the bioreactors according to the presentinvention can be considered as the next generation of migrationbioreactors that may move beyond a simple MicroTransWell (MTW) system toone that more closely replicates in vitro the microenvironment livingtissue.

Moreover, the application of microfabrication techniques, microfluidics,and microbiosensors with the bioreactors according to the presentinvention offers an opportunity for study of the molecular mechanism oftumor angiogenesis as well as leukocyte and cancer cell extravasation.For example, the systematic examination of the role of Tie2 and VEGF invascular formation and remodeling and may identify more specificmolecular targets for anti-angiogenic therapy. A similar microdevicemodel could be used to examine leukocyte and cancer cell extravasation.These devices will provide an appropriate cellular environment to hostmouse tumor explants, thereby potentially providing a metastasis assayfor tumor biopsy material. Metabolic sensing in these bioreactors willhelp provide a clearer understanding of the tumor microenvironment andconfirm the validity of our in vitro systems⁶²⁻⁶⁵.

Additionally, the limitation of the planar Borenstein design that thereis too little surface area of capillaries available to support thegrowth of a substantial volume of cells is overcome by the presentinvention, which remedies this problem by creating a multi-layerintercalated supply and return bioreactor that allows the full surfaceof a planar bioreactor to be covered with capillaries, and hencecapillary-perfused cells.

More specifically, in one aspect, the present invention relates tobioreactors. These bioreactors are biomicroelectromechanical systems(BioMEMS) that serve as migration microenvironments to study molecularmechanisms of tumor angiogenesis, tumor metastasis and leukocytemigration, but can also function as more general tissue bioreactors andperfusion systems. Among other things, one unique aspect of thesemicrofluidic devices is their integration of suitable cell culture andmicrofabrication techniques, which permit cell growth in small,confined, well-perfused volumes at tissue densities, provide independentcontrol of multiple chemokines and growth factor gradients, shearforces, tissue perfusion, and permeability of physical barriers tocellular migration, and allow detailed optical and electrochemicalobservation of normal and cancerous cells during cell migration,intravasation, extravasation, angiogenesis, and other cellularprocesses.

Recent advances in the fabrication of nanofilters⁵⁷⁻⁶¹ can be used topractice the present invention to provide perfused-membrane bioreactorsthat can allow the growth of mixed cultures of cells at near-to-tissuedensities in 1 mm×1 mm×100 micron volumes, in the presence ofcontrolled, stable chemokine or growth-factor gradients within thedevice, to mimic the in vivo tumor microenvironment. One advantage ofthe present invention is that custom devices can be constructed suchthat the isolated perfusion and cell-delivery systems allow independentcontrol of shear stress and chemokine gradients during the course of anexperiment. Moreover, the optical and electrochemical metabolicmicrosensors can be installed within these bioreactors to allowsimultaneous quantification of the local metabolic and chemicalenvironment (lactate, pH, O₂, etc.) in selected regions within thereactor, while cell migration or cell signaling events are imaged byfluorescence microscopy. Hence the next generation of migrationbioreactors will eventually move beyond a simple MicroTransWell (MTW)system to one that more closely replicates in vitro the microenvironmentliving tissue.

The application of microfabrication techniques, microfluidics, andmicrobiosensors offers an opportunity for study of the molecularmechanism of tumor angiogenesis as well as leukocyte and cancer cellextravasation. For example, the systematic examination of the role ofTie2 and VEGF in vascular formation and remodeling and may identify morespecific molecular targets for anti-angiogenic therapy. A similarmicrodevice model could be used to examine leukocyte and cancer cellextravasation. These bioreactors will provide an appropriate cellularenvironment to host mouse tumor explants, thereby potentially providinga metastasis assay for tumor biopsy material. Metabolic sensing in thesebioreactors will help provide a clearer understanding of the tumormicroenvironment and confirm the validity of our in vitro systems⁶²⁻⁶⁵.

Without intent to limit the scope of the invention, exemplary devices,application of them and related observations according to theembodiments of the present invention are given below. Note that titlesor subtitles may be used in the examples for convenience of a reader,which in no way should limit the scope of the invention. Moreover,certain theories may have been proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the devices and applications of them arepracticed according to the invention without regard for any particulartheory or scheme of action.

EXAMPLES Bioreactor with One Barrier

Referring now to FIGS. 1A and 1B, the present invention can be practicedin association with an inventive bioreactor 100 as shown in FIGS. 1A and1B. In one embodiment, the bioreactor 100 includes a first substrate 140having a first surface 140 a and an opposite second surface 104 b,defining a chamber 101 therebetween for receiving cells and a liquidmedium. The bioreactor 100 has a barrier 104 dividing the chamber 101into a first subchamber 102 and a second subchamber 103, wherein thebarrier 104 has a porosity to allow the first subchamber 102 and thesecond subchamber 103 in fluid communication and allow at least onepredetermined type of cells to permeate between the first subchamber 102and the second subchamber 103. The porosity of the barrier 104 can alsobe chosen not to let any cells to permeate.

As formed, the first subchamber 102 is adapted for receiving a firsttype of material such as cells 113 and the second subchamber 103 isadapted for receiving a second type of material such as cells 114,wherein each of the first type of material and the second type ofmaterial contains at least one selected from the group of cells,chemicals, and fluids. The cells can be any type of living cells,including, but not limited to, bacteria, protozoa, or both, normalcells, tumor cells, or any combination of them.

A biocompatible coating layer 116 can be applied to the chamber walls ofthe bioreactor 100, wherein the biocompatible coating layer 116 includesa material that may inhibit cell adhesion to the biocompatible coatinglayer, enhance cell adhesion to the biocompatible coating layer, orfunction as a fluorescent marker or indicator of the state of cells.

The bioreactor 100 further includes at least one or more inlet ports105, 106 and one or more corresponding input transfer channel 112, 107.As formed, the input transfer channel 112 is in fluid communication withthe corresponding inlet port 105 and the first subchamber 102, and theinput transfer channel 107 is in fluid communication with thecorresponding inlet port 106 and the second subchamber 103 for allowingdelivery of the cells, fluids or chemicals to the correspondingsubchamber 102 or 103, respectively. For example, a fluid can beintroduced from an external device (not shown) into the first subchamber102 through the inlet port 105 and the corresponding input transferchannel 112. Inlet ports 105, 106 each can be in fluid communicationwith an external device or port (not shown).

The bioreactor 100 additionally includes at least one or more outletports 111, 109 and one or more corresponding outlet transfer channel110, 108. As formed, the outlet transfer channel 110 is in fluidcommunication with the corresponding outlet port 111 and the firstsubchamber 102, and the outlet transfer channel 108 is in fluidcommunication with the corresponding outlet port 109 and secondsubchamber 103 for allowing removal of the cells, fluids or chemicalsfrom the corresponding subchamber 102 or 103, respectively. For example,a fluid can be introduced away from the first subchamber 102 through theoutlet transfer channel 110 and the corresponding outlet port 111.Outlet ports 111, 109 each can be in fluid communication with anexternal device or port (not shown).

The bioreactor 100 further includes at least one or more auxiliary ports115 and one or more auxiliary channels 115 a. As formed, each auxiliarychannel 115 a is in fluid communication with a corresponding auxiliaryport 115 and a corresponding one of the input transfer channels 112, 107and the outlet transfer channels 110, 108 for flushing the correspondingtransfer channel. Auxiliary ports 115 each can be in fluid communicationwith an external device or port (not shown). Auxiliary ports 115 andauxiliary channels 115 a can be utilized to prevent clogging by cells orcellular debris in the bioreactor 100. They can also be utilized toselectively apply coatings to the channels to which they are in fluidcommunication.

The bioreactor 100 additionally includes one or more access ports 117and one or more access channels 117 a. As formed, each access channel117 a is in fluid communication with a corresponding access port 117 anda corresponding one of the first subchamber 102 and the secondsubchamber 103 for allowing delivery or removal of the cells, fluids,chemicals, coating material or sensing material to the correspondingsubchamber. The access ports 117 and corresponding access channels 117 aare strategically positioned so as to provide direct access to the firstsubchamber 102 and the second subchamber 103. For example, a fluid canbe introduced into the first subchamber 102 through an access channels117 a and the corresponding access port 117 fast because the distancebetween the access port 117 and the first subchamber 102 is the shortestfor this embodiment. Each access port 117 can be in fluid communicationwith an external device or port (not shown).

Moreover, the bioreactor 100 has a second substrate 150, wherein thesecond substrate 150 is positioned adjacent to the first surface 140 aof the first substrate 140 and defines a plurality of connectionchannels 155. Each of the connection channels 155 is formed so as to bein fluid communication with a corresponding one of the inlet ports 105,106, the outlet ports 111, 109, the auxiliary ports 115, and the accessports 117 as set forth above.

The bioreactor 100 further includes a plurality of connection ports 151corresponding to the plurality of connection channels 155. Each of theconnection ports 151 is formed with a channel 153 and is strategicallypositioned to the second substrate 150 such that each channel 153 of theconnection ports 151 is in fluid communication with a corresponding oneof the connection channels 155 formed in the second substrate 150 asshown in FIG. 1B.

The first substrate 140 can be fabricated from glass, Mylar, PDMS,silicon, a polymer, a semiconductor, or any combination of them. Thebarrier 104 is formed with a porous material. The barrier 104 can bemicrofabricated so as to form a structure allowing the fluidcommunication between the first subchamber 102 and the second subchamber103, which may allow permeation of the barrier 104 by certainpredetermined types of cells but not by other types of cells. Forexample, in the embodiment shown in FIGS. 1A and 1B, the barrier 104 isformed with a plurality of posts spaced from each other so as to allowbacteria to cross over but not protozoa.

The bioreactor 100 further has a third substrate 160, which ispositioned adjacent to the first surface of the first substrate 140, andmeans strategically positioned in the third substrate 160 and adaptedfor electrochemical measurements of the cells responsive to the liquidmedium in one or both of the first subchamber 102 and the secondsubchamber 103. The third substrate 160 can be formed with asemiconductor material such as silicon.

In one embodiment as shown in FIG. 1B, the means for electrochemicalmeasurements includes a reference electrode 161, a counter electrode162, a plurality of edge connector pads 164, and a plurality ofelectrically conductive leads 163. A first electrically conductive lead163 electrically couples the reference electrode 161 to a correspondingedge connector pad 164, and a second electrically conductive lead 163electrically couples the counter electrode 162 to a corresponding edgeconnector pad 164. The means for electrochemical measurements furtherincludes a plurality of individually addressable working electrodes 165.Each of the plurality of individually addressable working electrodes 165is electrically coupled to a corresponding edge connector pad 164through a corresponding electrically conductive lead 163. The sensingheads of the plurality of individually addressable working electrodes165 are strategically positioned in a region shown by outline 166 inFIG. 1B.

In operation, the liquid medium being introduced into one or both of thefirst subchamber 102 and the second subchamber 103 may include one ormore analytes, and the plurality of individually addressable workingelectrodes are adapted for sensing the concentration of a single analyteof the liquid medium at multiple locations in the chamber 101 or theconcentrations of a plurality of analytes of the liquid medium atmultiple locations in the chamber 101 at a time period shorter than acharacteristic reaction time related to at least one of cellularphysiological activities of the cells. The plurality of individuallyaddressable working electrodes can be further adapted to be capable ofmeasuring the metabolic variables related to the cells responsive to theliquid medium at multiple locations in the chamber 101 at a time periodshorter than a characteristic reaction time related to at least one ofcellular physiological activities of the cells. The sensing heads of theplurality of individually addressable working electrodes 165 arestrategically positioned in a region shown by outline 166 correspondingto that of the chamber 101 to perform such tasks.

The bioreactor 100 further includes a fourth substrate 170, wherein thefourth substrate 170 is positioned above the second surface 140 b of thefirst substrate 140, and means strategically positioned in the fourthsubstrate 170 and adapted for optical measurements of the cellsresponsive to the liquid medium in at least one of the first subchamber102 and the second subchamber 103. The fourth substrate 170 is at leastpartially transparent. For examples, it can be formed with asemiconductor material or a glass or both.

In one embodiment as shown in FIG. 1B, the means for opticalmeasurements includes a plurality of optical sensors 171, a plurality ofedge connector pads 173, and a plurality of leads 172, each coupling anoptical sensor 171 to a corresponding edge connector pad 173. Theplurality of optical sensors 171 may include at least one deviceselected from the group of an LED and photodiode pair, a fiber opticcoupler, and an optical detecting head.

In operation, the liquid medium being introduced into one or both of thefirst subchamber 102 and the second subchamber 103 may include one ormore analytes, and the plurality of optical sensors 171 are adapted tobe capable of sensing the concentration of a single analyte of theliquid medium at multiple locations in the chamber 101 or theconcentrations of a plurality of analytes of the liquid medium atmultiple locations in the chamber 101 at a time period shorter than acharacteristic reaction time related to at least one of cellularphysiological activities of the cells. The plurality of optical sensors171 can be further adapted for measuring the metabolic variables relatedto the cells responsive to the liquid medium at multiple locations inthe chamber 101 at a time period shorter than the characteristicreaction time related to at least one of cellular physiologicalactivities of the cells. The sensing heads of the plurality of opticalsensors 171 are strategically positioned in a region shown by outline174 corresponding to that of the chamber 101 to perform such tasks.

Bioreactor with Multiple Barriers

Referring now to FIG. 2, the present invention can also be practiced inassociation with an inventive bioreactor 700 as shown in FIG. 2. In oneembodiment, the bioreactor 700 includes a substrate 730 having a firstsurface and an opposite second surface, defining a chamber 732therebetween for receiving cells and a liquid medium, wherein thechamber 732 is formed with a center 734 and a boundary 736. Thebioreactor 700 also has a first barrier 738, which encloses the center734 and a portion of the chamber 732 to form a central chamber 706, anda second barrier 740, which is positioned between the first barrier 738and the boundary 736 so as to form an intermediate chamber 705 and anouter chamber 704.

In one embodiment, the first barrier 738 has a first porosity to allowthe central chamber 706 and the intermediate chamber 705 in fluidcommunication and allow at least a first predetermined type of cells topermeate between the central chamber 706 and the intermediate chamber705, and the second barrier 740 has a second porosity to allow the outerchamber 704 and the intermediate chamber 705 in fluid communication andallow at least a second predetermined type of cells to permeate betweenthe outer chamber 704 and the intermediate chamber 705.

Moreover, the central chamber 706 is adapted for receiving a first typeof material such as tumor cells 714, the intermediate chamber 705 isadapted for receiving a second type of material such as normal tissuecells 713, and the outer chamber 704 is adapted for receiving a thirdtype of material such as endothelial cells 712. Each of the first typeof material, the second type of material and the third type of materialcontains at least one selected from the group of cells, chemicals, andfluids.

The first predetermined type of cells includes tumor cells 714, whichnormally is received in the central chamber 706 that is formed tosimulate a tumor space. The second predetermined type of cells includesnormal tissue cells 713, which normally is received in the intermediatechamber 705 that is formed to simulate a tissue space. Furthermore, theouter chamber 704 is formed to simulate a vascular space adapted forreceiving endothelial cells, macrophage cells, neutophil cells, anycombination of them, or other immune cell type.

A biocompatible coating layer 742 can be applied to the chamber walls atthe boundary 736, wherein the biocompatible coating layer 742 includes amaterial that may inhibit cell adhesion to the biocompatible coatinglayer, enhance cell adhesion to the biocompatible coating layer, orfunction as a fluorescent marker or indicator of the state of cells.

The bioreactor 700 further includes one or more inlet or outlet ports701 and corresponding one or more input or output transfer channels 751,where each of the input or output transfer channel 751 is in fluidcommunication with a corresponding inlet or outlet port 701 and theouter chamber 704 for allowing delivery of cells, fluids or chemicals tothe outer chamber 704.

The bioreactor 700 additionally may include one or more inlet or outletports 702 and corresponding one or more input or output transferchannels 752, where each of the input or output transfer channels 752 isin fluid communication with a corresponding inlet or outlet port 702 andthe central chamber 706 for allowing delivery of the cells, fluids orchemicals to the central chamber 706.

The bioreactor 700 may further include one or more inlet or outlet ports703 and corresponding one or more input or output transfer channels 753,where each of the input or output transfer channels 753 is in fluidcommunication with a corresponding inlet or outlet port 703 and theintermediate chamber 705 for allowing delivery of the cells, fluids orchemicals to the intermediate chamber 705.

The substrate 730 can be fabricated from glass, Mylar, PDMS, silicon, apolymer, a semiconductor, or any combination of them. The first barrier738 is formed with a porous material. The first barrier 738 can bemicrofabricated so as to form a first structure allowing the fluidcommunication between the central chamber 706 and the intermediatechamber 705. The second barrier 740 is formed with a porous material.The second barrier 740 can be microfabricated so as to form a secondstructure allowing the fluid communication between the outer chamber 704and the intermediate chamber 705. The first barrier 738 and the secondbarrier 740 can be formed with same or different porous materials. Andthe second structure can be same or different from the first structure.For example, in the embodiment shown in FIG. 2, the first barrier 738 isformed with a plurality of posts spaced from each other more condensedthan the second barrier 740. The first barrier 738 and the secondbarrier 740 can also be formed into same or different shapes. Forexample, in the embodiment shown in FIG. 2, the first barrier 738 andthe second barrier 740 are substantially circular. The boundary 736 cantake various geometric shapes as well. For example, in the embodimentshown in FIG. 2, the boundary 736 is substantially circular.

The bioreactor 700 further includes means strategically positioned andadapted for electrochemical measurements of the cells responsive to theliquid medium in one or more of the outer chamber 704, the intermediatechamber 705 and the central chamber 706.

In one embodiment as shown in FIG. 2, the means for electrochemicalmeasurements includes a reference electrode 707, a counter electrode708, and a plurality of individually addressable working electrodes.

In operation, the liquid medium being introduced into one or more of theouter chamber 704, the intermediate chamber 705 and the central chamber706 may include one or more analytes, and the plurality of individuallyaddressable working electrodes include a first group of individuallyaddressable working electrodes 709, a second group of individuallyaddressable working electrodes 710 and a third group of individuallyaddressable working electrodes 711, respectively.

For the embodiment shown in FIG. 2, the first group of individuallyaddressable working electrodes 709 are adapted to be capable of sensingthe concentration of a single analyte of the liquid medium at multiplelocations in the outer chamber 704 or the concentrations of a pluralityof analytes of the liquid medium at multiple locations in the outerchamber 704 at a time period shorter than a characteristic reaction timerelated to at least one of cellular physiological activities of thecells. The first group of individually addressable working electrodes709 are further adapted to be capable of measuring the metabolicvariables related to the cells responsive to the liquid medium atmultiple locations in the outer chamber 704 at a time period shorterthan a characteristic reaction time related to at least one of cellularphysiological activities of the cells.

The second group of individually addressable working electrodes 710adapted to be capable of sensing the concentration of a single analyteof the liquid medium at multiple locations in the central chamber 706 orthe concentrations of a plurality of analytes of the liquid medium atmultiple locations in the central chamber 706 at a time period shorterthan a characteristic reaction time related to at least one of cellularphysiological activities of the cells. The second group of individuallyaddressable working electrodes 710 are further adapted to be capable ofmeasuring the metabolic variables related to the cells responsive to theliquid medium at multiple locations in the central chamber 706 at a timeperiod shorter than a characteristic reaction time related to at leastone of cellular physiological activities of the cells.

Similarly, the third group of individually addressable workingelectrodes 711 are adapted to be capable of sensing the concentration ofa single analyte of the liquid medium at multiple locations in theintermediate chamber 705 or the concentrations of a plurality ofanalytes of the liquid medium at multiple locations in the intermediatechamber 705 at a time period shorter than a characteristic reaction timerelated to at least one of cellular physiological activities of thecells. The third group of individually addressable working electrodes711 are further adapted to be capable of measuring the metabolicvariables related to the cells responsive to the liquid medium atmultiple locations in the intermediate chamber 705 at a time periodshorter than a characteristic reaction time related to at least one ofcellular physiological activities of the cells.

As such formed, among other things, bioreactor 700 can be utilized tonurture, culture, observe, detect and explore cells, collection ofcells, biofilm formed by cells and related cell activities. Forexamples, as shown in FIG. 2, bioreactor 700 allows a spectrum of cellactivities to take place, including: a cell 715, which can be an immunetype of cell such as a macrophage or neutophil, undergoing extravasationacross the second barrier 740 from the outer chamber 704 into theintermediate chamber 705, a cell 716, which can be a tumor cellmetastasizing from the central chamber 706 through the surroundingtissue into the vascular space, undergoing intravasation across thesecond barrier 740 from the intermediate chamber 705 into the outerchamber 704, and a cell 717, for example, an endothelial cell,undergoing tube formation across the second barrier 740 that mayeventually lead to vascularization of the tumor, respectively.

Bioreactors with an Array of Chambers and a Common Feed Line

Referring now to FIGS. 2-6, the present invention can be practiced inassociation with an inventive bioreactor 200 as shown in FIGS. 2-6. Inone embodiment, referring first to FIGS. 2A and 2B, the bioreactor 200includes a substrate 230 having a first surface and an opposite secondsurface. The bioreactor 200 has a plurality of array of chambers 204formed on the substrate 230. Each array of chambers 204 is adapted forreceiving cells in a liquid medium and includes a channel 202 and aplurality of chambers 206 formed in the substrate 230. Each of theplurality of chambers 206 is adapted for receiving cells in a liquidmedium and formed with an open end 262, an opposite closed end 264 andsidewalls 266. The open end 262 and the closed end 264 of a particularchamber 206 define a depth, d, therebetween for the correspondingchamber 206, which is in fluid communication with the channel 202through the open end 262. Additionally, the sidewalls 266 defines awidth, w, therebetween for the corresponding chamber 206. As formed, atleast two of the plurality of chambers 206, either from same array ordifferent arrays, may have depths or widths same or different from eachother. This design allows the bioreactor 200 to provide a variety ofenvironments to cells tailored for different applications. The substrate230 can be fabricated from glass, Mylar, PDMS, silicon, a polymer, asemiconductor, or any combination of them.

Each array of chambers 204 may further includes an inlet port 201 formedin fluid communication with the channel 202, and an outlet port 203formed in fluid communication with the channel 202, wherein the inletport 201 and the outlet port 203 are apart from each other along thechannel 202. As such formed, a fluid or an intended amount of materialsuch as a bolus of selected chemicals 206 can be introduced from anexternal device or port (not shown) into the channel 202 through theinlet port 201, and away from the channel 202 through the outlet port203. Thus, to each array 204, channel 202 serves as a common feed lineto the plurality of chambers 206.

Each of the plurality of chambers 206 is adapted to receive and cultureat least one predetermined type of cells. The cells can be any type ofliving cells, including, but not limited to, bacteria, protozoa, orboth, normal cells, tumor cells, or any combination of them. Cells canbe introduced into a chamber individually, in a collection of cells, orin the form of biofilms. Different chambers can have same or differenttypes of cells.

The bioreactor 200 further includes a barrier 209 for at least one ofthe chambers 206, wherein the barrier 209 is positioned substantially atthe open end 262 of a corresponding chamber 206 as shown in FIG. 2B. Thebarrier 209 has a porosity to allow the corresponding chamber 206 andthe channel 202 in fluid communication to each other. The barrier 209also allows at least one predetermined type of cells to permeate betweenthe corresponding chamber 206 and the channel 202 and at least anotherpredetermined type of cells not to permeate between the correspondingchamber 206 and the channel 202. The barrier 209 can also allows nocells to permeate at all. Thus, the barrier 209 has a selective porosityfor the cells and functions as a filter as well.

The bioreactor 200 also includes a biocompatible coating layer 205applied to the channel walls, wherein the biocompatible coating layer205 comprises a material that may inhibit cell adhesion to thebiocompatible coating layer to keep the fluid communication in thechannel 202 open. Alternatively, in place of the biocompatible coatinglayer 205, other means such as a leaky light guide can be utilized.

The bioreactor 200 may further include a biocompatible coating layer 207applied to the sidewalls 266 of a chamber 206. The biocompatible coatinglayer 207 comprises a material that may inhibit cell adhesion to thebiocompatible coating layer 207, enhance cell adhesion to thebiocompatible coating layer 207, or function as a fluorescent marker orindicator of the state of cells. Different chambers 206 may have same ordifferent coating layers.

The bioreactor 200 further includes at least one or more auxiliary ports214 and corresponding auxiliary channels 214 a. As formed, an auxiliarychannel 214 a is in fluid communication with a corresponding auxiliaryport 214 and a corresponding chamber 206 for allowing individual controlof the environment of the corresponding chamber 206. The individualcontrol of the environment of the corresponding chamber 206 includes anyand all intended activities that may affect the environment of a chambersuch as the delivery or removal of the cells, fluids or chemicals to thecorresponding chamber 206 or flushing the corresponding chamber 206.

In an alternative embodiment as shown in FIG. 3, a bioreactor 300includes at least one sample chamber 302 and a plurality of samplechannels 301, wherein the plurality of sample channels 301 are in fluidcommunication with the sample chamber 302 and a corresponding chamber306. As formed, the sample chamber 302 is in fluid communication with atleast one corresponding auxiliary channel 383 that is in fluidcommunication with at least one corresponding auxiliary port 303, forallowing individual control of the environment of the correspondingsample chamber 302. The individual control of the environment of thecorresponding sample chamber 302 includes any and all intendedactivities that may affect the environment of a sample chamber 302 suchas the delivery or removal of the fluids, or materials, or substancesuch as chemicals to the corresponding sample chamber 302. The samplechamber 302 is further adapted for receiving a sample 380 of hostmaterial, such as soil, that provides exudates affecting the cells orbiofilm in the corresponding chamber 306. The sample chamber 302 is alsoformed with a closed end 382 and an opposite open end 384 through whichthe host material can be received into or removed from the samplechamber 302. Additionally, a lid (not shown) adapted for slidablycovering or opening the open end 384 of the sample chamber 302 can beutilized. This type of the bioreactor according to the embodiment of thepresent invention in FIG. 3 allows one to, among other things, observe,detect, adjust, control, and/or utilize the effects of exudates from asample of host material on the cells growing in the chamber of thebioreactor.

In another alternative embodiment as shown in FIG. 5, a bioreactor 500includes at least one sample chamber 502 that is formed in fluidcommunication with a corresponding chamber 506. The sample chamber 502is adapted for receiving a sample of host material 501 that can directlyaffect the cells or biofilm in the corresponding chamber 506 because thesample chamber 502 is directly in fluid communication with acorresponding chamber 506. The sample chamber 502 is formed with aclosed end 582 and an opposite open end 584 through which the sample ofhost material 501 can be received into or removed from the samplechamber 502. Additionally, a lid (not shown) adapted for slidablycovering or opening the open end 584 of the sample chamber 502 can beutilized. This type of the bioreactor according to the embodiment of thepresent invention in FIG. 5 allows one to, among other things, observe,detect, adjust, control, and/or utilize the effects of a sample of hostmaterial on the cells growing in the chamber of the bioreactor.

The bioreactor 200 additionally includes, referring now to FIGS. 2A and2B, means adapted for electrochemical measurements of the cellsresponsive to the liquid medium in at least one of the chambers 206. Inone embodiment, the means for electrochemical measurements includes acounter electrode 211, a reference electrode 212, and a plurality ofelectrically conductive leads. Among the plurality of electricallyconductive leads, a first electrically conductive lead 272 aelectrically couples the reference electrode 212 to a corresponding edgeconnector pad (not shown), and a second electrically conductive lead 272b electrically couples the counter electrode 211 to a corresponding edgeconnector pad (not shown). The means for electrochemical measurementscan also be used to measure the electrochemical constituents outside thecells that reflect the status of the cells, the culture medium, or thecellular exudates.

The means for electrochemical measurements further includes a pluralityof individually addressable working electrodes 270 and a plurality ofcorresponding amplifiers 210. Each individually addressable workingelectrode 270 is electrically coupled to a corresponding amplifier 210through a corresponding electrically conductive lead 272. The bioreactor200 further includes a plurality of electrically conductive output leads274, each electrically coupling a corresponding amplifier 210 to anoutput device such as a multiplexed potentiostat (not shown).

In operation, the liquid medium being introduced into an array 204through a corresponding channel 202 (and into one or more chambers 206)may include one or more analytes, and the plurality of individuallyaddressable working electrodes 270 are adapted for capable of sensingthe concentration of a single analyte of the liquid medium at multiplelocations in a corresponding chamber 206 or the concentrations of aplurality of analytes of the liquid medium at multiple locations in thecorresponding chamber 206 at a time period shorter than a characteristicreaction time related to at least one of cellular physiologicalactivities of the cells. The plurality of individually addressableworking electrodes 270 are further adapted for capable of measuring themetabolic variables related to the cells responsive to the liquid mediumat multiple locations in the corresponding chamber 206 at a time periodshorter than a characteristic reaction time related to at least one ofcellular physiological activities of the cells.

Alternatively, as shown in FIG. 4, a bioreactor 400 has a plurality ofcontrolling ports 404 and a plurality of connection channels 494,wherein each of the connection channels 494 is in fluid communicationwith a corresponding controlling port 404 and a chamber 496. Thebioreactor 400 further includes a fluid control valve 402 adapted forcontrolling the fluid communication between the plurality of controllingports 404 and the chamber 496, wherein the fluid control valve 402includes a pneumatic or mechanical valve. A control port 401 adapted forcontrolling the fluid control valve 402 can also be provided.

In this embodiment, the counter electrode 405 and the referenceelectrode 406 are positioned between the fluid control valve 402 and theplurality of controlling ports 404, wherein the liquid medium includesat least one or more analytes, and wherein the plurality of individuallyaddressable working electrodes are positioned between the fluid controlvalve 402 and the plurality of controlling ports 404 and adapted forcapable of sensing the concentration of a single analyte of the liquidmedium corresponding to multiple locations in a corresponding chamber496 or the concentrations of a plurality of analytes of the liquidmedium corresponding to multiple locations in the corresponding chamber496 at a time period shorter than a characteristic reaction time relatedto at least one of cellular physiological activities of the cells. Theplurality of individually addressable working electrodes are furtheradapted for capable of measuring the metabolic variables related to thecells responsive to the liquid medium at multiple locationscorresponding to the corresponding chamber 496 at a time period shorterthan a characteristic reaction time related to at least one of cellularphysiological activities of the cells. This type of the bioreactoraccording to the embodiment of the present invention in FIG. 4 allowsone to, among other things, minimize disturbances such as biofouling ofthe sensors to the cells in the chamber because the sensors arephysically separated from the chamber of the bioreactor.

In another embodiment as shown in FIG. 6, the reference electrode 612can be strategically positioned as a common reference electrode andadapted for electrochemical measurements of the cells responsive to theliquid medium in the plurality of chambers. Correspondingly, in each ofthe plurality of chambers, a counter electrode is adapted forelectrochemical measurements of the cells responsive to the liquidmedium in a corresponding chamber to allow the plurality of chambers tobe operated individually and the means for electrochemical measurementsfor the plurality of chambers to be activated for one or more chambersat a time sequentially. For examples, a counter electrode 611 is adaptedfor electrochemical measurements of the cells responsive to the liquidmedium in a corresponding chamber 606 a, and a counter electrode 613 isadapted for electrochemical measurements of the cells responsive to theliquid medium in a corresponding chamber 606 a, respectively.

The bioreactor of this invention further includes means positioned inthe channel and adapted for monitoring of the cells therein optically,electrically or both. In one embodiment as shown in FIGS. 2A and 2B, themeans for monitoring of the cells can include at least one opticalsensor 213 and at least one lead 276 in optical communication with acorresponding optical sensor 213. The optical sensor 213 includes atleast one device selected from the group of an LED and photodiode pair,a fiber optic coupler, and an optical detecting head. Other opticaldevices can be utilized as well. Alternatively, the means for monitoringof the cells includes at least one electrical sensor 213 and at leastone lead 276 in electrical communication with a corresponding electricalsensor 213. Such monitoring means can also be utilized to monitor otherdynamic activities in the channel, for example, activities and responsesof cells when a bolus 208 of selected chemicals moves along the channel202, which is adapted for allowing such movement of material along thechannel 202.

The bioreactors of this invention can find many applications. Inaddition to applications set forth elsewhere and among other things,they can be utilized for culturing, studying and observing a pluralityof biofilms simultaneously, where each biofilm may contain apredetermined type of cells that are same or different from otherbiofilms. Each array can receive one or more collection of cells in oneor more chambers to grow. A bolus of selected chemicals or othersubstances, same or different for different arrays, can be introduced tomove along a corresponding channel for each array of chambers. And aspectrum of dynamic properties due to the interfacing between the cellsand the bolus of selected chemicals or other substances can be observed,detected, collected, analyzed and utilized.

Capillary Bioreactor

Referring now to FIGS. 11(A-D), the present invention can be practicedin association with an inventive bioreactor 1100 and its variants asshown in FIGS. 11(A-D). In one embodiment, referring first to FIGS. 11A,11A2 and 11A3, the bioreactor 1100 includes a first substrate 1124having a first surface 1124 a, an opposite second surface 1124 b andedges. The bioreactor 1100 further includes a second substrate 1121having a first surface 1121 a and an opposite second surface 1121 b,defining a cavity 1121 c with a bottom surface 1121 d, where the bottomsurface 1121 d is located therebetween the first surface 1121 a and thesecond surface 1121 b. The first surface 1124 a of the first substrate1124 is received by the second surface 1121 b of the second substrate1121 to cover the cavity 1121 c so as to form a channel 1101 forreceiving cells and a liquid medium. The second substrate 1121 can befabricated from glass, Mylar, PDMS, silicon, a polymer, a semiconductor,or any combination of them. The first substrate 1124 is at leastpartially optically transparent such that the dynamic activities ofcells in the channel 1101 are detectable through optical detectingmeans.

A recess 1105 c is formed in the second substrate 1121 with a bottomsurface 1105 d and in fluid communication with the channel 1101.Additionally, a barrier 1106 is positioned for covering the recess 1105c so as to form an outer chamber 1105. The barrier 1006 has a porosityto allow the channel 1101 and the outer chamber 1105 to be in fluidcommunication and control the move of at least one predetermined type ofcells between the channel 1101 and the outer chamber 1105. In oneembodiment as best shown in FIG. 11A 1, the barrier 1106 includes aplurality of posts spaced with a gap from each other. These posts may becoated in certain locations with substances to prevent entry of cells,particularly the endothelial cells. Gaps between the posts in certainlocations allow for delivery of particular cell types to the outerchamber 1105The second substrate 1121 further defines a first opening1101 a and an opposite, second opening 1101 b adapted for allowing aflow of liquid to be introduced into the channel 1101 through the firstopening 1101 a and away from the channel 1101 through the second opening1101 b substantially along a first direction 1101 c.

The bioreactor 1100 further includes a biocompatible coating layer 1102applied to the interior surfaces of the second substrate 1121 around thechannel 1101. The biocompatible coating layer 1102 includes a materialthat may inhibit cell adhesion to the biocompatible coating layer,enhance cell adhesion to the biocompatible coating layer, promoteorganization and growth of cells, or function as a fluorescent marker orindicator of the state of cells.

In forming the bioreactor 1100, the channel 1101 is sized to allow thegrowth of a layer of cells 1103 on the biocompatible coating layer 1102and the flow of liquid in the channel 1101. The flow of liquid iscontrolled so as to provide a known shear force to the layer of cells1103. The flow of liquid can be further controlled so as to provide anenvironment that simulates a vascular space in the channel 1101. Forexamples, the channel 1101 can be used for introduction of endothelialcells, and for their subsequent perfusion.

The cells can be any type of living cells, including, but not limitedto, bacteria, protozoa, or both, normal cells, endothelial cells, tumorcells, or any combination of them. Cells can be introduced into thechannel 1101 individually, in a collection of cells, or in the form ofbiofilm. In one embodiment, the layer of cells 1103 substantially formsan endothelial cells lined capillary in the channel 1101. The channel1101 is sized such that when at least one cell 1104 that is not one ofthe endothelial cells, such as a tumor cell, is introduced into thechannel 1101, it can undergo intravasation in the endothelial cellslined capillary.

In an alternative embodiment as shown in FIG. 11C, the second substrate1121 further defines one or more injection ports 1140, 1141 in fluidcommunication with the channel 1101 to allow a stream of substance to beintroduced into the channel 1101 through the injection port 1140, 1141substantially along a second direction 1148, respectively. As shown inFIG. 11C, the second direction 1148 is substantially perpendicular tothe first direction 1101 c. The stream of substance is controlled so asto provide a gradient to the channel 1101. The stream of substanceincludes a substance affecting the growth of cells such as chemokine.

Referring now to FIG. 11A 1, 11A2 and 11A3, the outer chamber 1105 issized to allow the growth of a host of cells. The host of cells includesat least a first type of cells 1107 and a second type of cells 1113 thatis different from the first type of cells 1107. In other words, theouter chamber 1105 is sized to allow the growth of two types of cells.In one embodiment, the first type of cells 1107 includes normal cells,and the second type of cells 1113 includes tumor cells. These host cellscan either be grown in the outer chamber 1105, or so as to avoidundesired growth of endothelial cells into the host cell population,they may be grown on a suitable substrate outside of the bioreactor 1100and then be introduced as a complete unit or smaller units into theouter chamber 1105 either through one of the microfluid ports or bytemporary removal of the fist substrate 1124 such as a glass lid of theouter chamber 1105. As best shown in FIG. 11A 2, a port 1122 and aconnection channel 1123 are formed in the second substrate 1121 suchthat the connection channel 1123 is in fluid communication with theouter chamber 1105 and the port 1122. The bioreactor 1100 furtherincludes a plurality of electrodes 1114, 1115, 1116 adapted forelectrochemical measurements of the host of cells. Moreover, thebioreactor 1100 further includes a plurality of controlling ports 1108,1109, 1110 and a plurality of connection channels 1108 a, 1109 a, 1110a, wherein each of the connection channels 1108 a, 1109 a, 1110 a is influid communication with a corresponding one of controlling ports 1108,1109, 1110 and the outer chamber 1105, respectively.

Bioreactor 1100 and its variants as given above can find manyapplications. Such a readily fabricated device is suitable for eventsthat are high-probability and in relatively short lengths of capillary,such as neutrophil binding to a tube of activated endothelial cells.Multiple channels/ports allow delivery of different cells, andestablishment of chemokine, nutrient, and pH gradients. Electrodesmeasure metabolism. Hence it may best suited for experiments orapplications involving activated or inflamed endothelial cells.

For example, as shown in FIG. 11B, bioreactor 1100 is used as acapillary perfused migration bioreactor for the study of cancer cell orneutrophil extravasation in the presence of two competing chemokinegradients. In particular, a first stream 1130 a of chemokine is injectedthrough a port 1130 and hence creates a first gradient of that chemokineboth in the outer chamber 1105 and the channel 1101. Additionally, asecond stream 1131 a of chemokine is injected through a port 1131 andhence creates a second gradient of that chemokine both in the outerchamber 1105 and the channel 1101. A spectrum of dynamics of the cellsmay happen. For instances, a cancer cell or a neutrophil 1132 movesalong the channel 1101 in response to either perfusion flow or thepresence of the first and second chemokine gradients, and a cancer cellor neutrophil 1133 undergoes extravasation from the channel 1101 intothe outer chamber 1105.

FIG. 11D illustrates another application. Here bioreactor 1100 is usedas a capillary perfused migration bioreactor for the study endothelialtube formation triggered by chemokine excreted by tumor cells, where theouter chamber 1105 of sufficient size contain a sufficiently largervolume of host cells 1108 that is required to ensure endothelial tubeformation. An endothelial tube 1140 is formed in response to eitherchemokine being released by the tumor cells 1113, or by microfluidicdelivery of vascular endothelial growth factor (VEGF) or othersubstances through the microfluidic port 1111. Note that another tumorcell 1112 is on the move. The electrodes 1114 may be able to detectchanges in the extracellular environment associated with tube formationand the associated proteolysis.

Bioreactor with Multiple Traps

Referring now to FIGS. 10(A-I), the present invention can also bepracticed in association with an inventive bioreactor 1000 and itsvariants as shown in FIGS. 10(A-I). In one embodiment, referring firstto FIGS. 10A, 10B, 10G, 10H and 10I, the bioreactor 1000 includes afirst substrate 1001 having a first surface 1001 a and an oppositesecond surface 1011 b, defining a chamber 1006 therebetween forreceiving cells 1008 and a liquid medium. The first substrate 1001 canbe fabricated from glass, Mylar, PDMS, silicon, a polymer, asemiconductor, or any combination of them.

An inlet port 1021 and a first connection channel 1021 a are formed inthe first substrate 1001, where the first connection channel 1021 a isin fluid communication with the inlet port 1021 and the chamber 1006 forallowing a stream of substance to be delivered to the chamber 1006.Additionally, an outlet port 1005 and a second connection channel 1005 aare formed in the first substrate 1001, where the second connectionchannel 1005 a is in fluid communication with the outlet port 1005 andthe chamber 1006 for allowing a stream of substance to be removed fromthe chamber 1006.

Moreover, as best shown in the insert of FIG. 10A, the bioreactor 1000has confining means 1003 positioned in a region in the chamber 1006proximate to the first connection channel 1021 a to confine the cells1008. In one embodiment, the confining means 1003 includes a pluralityof traps 1007, where each of the plurality of traps 1007 is capable ofreceiving at least one cell or a collection of cells 1008. Each of theplurality of traps 1007 includes a structure defining a recess 1007 a soas to receive and confine one or more cells 1008 therein. The structuremay be partially formed with a filter 1007 b to allow the recess 1007 ato be in fluid communication with the chamber 1006. The filter 1007 bcan be formed with a plurality posts spacing from each other with a gapg. Traps can take various shapes and have different physics properties.For examples, in the embodiment as shown in FIG. 10G, a trap 1090 hasdistinct posts and sides. In the embodiment as shown in FIG. 10H, a trap1092 has posts and sides that are extending to the posts. And in theembodiment as shown in FIG. 10I, a trap 1093 is formed with a singlepost and sides. The plurality of traps 1007 can be arranged to form anarray.

Additionally, the first substrate 1001 defines a first alternate port1022 and a third connection channel 1022 a in fluid communication withthe first alternate port 1022 and the first connection channel 1021 afor allowing additional substance to be introduced into the chamber1006. Moreover, the first substrate 1001 further defines a secondalternate port 1011, a third connection channel 1011 a, and a secondchamber 1009, wherein the third connection channel 1011 a is in fluidcommunication with second alternate port 1011 and the second chamber1009, and the second chamber 1009 is in fluid communication with thefirst connection channel 1021 a. Furthermore, the second chamber 1009 isformed with an oxygen permeable structure to provide oxygen to thecells.

Referring now to FIG. 10B, the bioreactor 1100 further includes a secondsubstrate 1050 having a first surface 1050 a and an opposite, secondsurface 1050 b, and means adapted for electrochemical measurements ofthe cells in the chamber 1006. The means for electrochemicalmeasurements is positioned with the second substrate 1050 such that whenthe first surface 1050 a of the second substrate 1050 is received by thesecond surface 1001 b of the first substrate 1001, the means forelectrochemical measurements is at a corresponding measurement position.The means for electrochemical measurements includes at least oneelectrode 1051 monitoring entry of the cells into the chamber 1006, atleast one electrode 1052 monitoring leaving of the cells from thechamber 1006, and a plurality of electrodes 1053 detecting chemicalspecies in the chamber 1006.

The bioreactor 1000 further includes a third substrate 1060 having afirst surface 1060 a and an opposite, second surface 1060 b, and meansadapted for optical measurements. The means for optical measurements ispositioned with the third substrate 1060 such that when the firstsurface 1060 a of the third substrate 1060 is received by the secondsurface 1001 b of the first substrate 1001, the means for opticalmeasurements is at a corresponding measurement position. The means foroptical measurements includes a plurality of optical sensors 1061strategically positioned for detecting chemical and biological specieswithin the chamber 1006 and the physiological state of the cells withinthe chamber 1006. The third substrate 1060 is at least partiallytransparent.

Bioreactor with Confined Region

Referring now to FIGS. 10(C-F), the present invention can also bepracticed in association with an inventive bioreactor 1000 and itsvariants as shown in FIGS. 10(C-F). In one embodiment, referring firstto FIGS. 10C, 10D, 10E and 10F, the bioreactor 1000 includes a firstsubstrate 1001 having a first surface 1001 a and an opposite secondsurface 1001 b, defining a chamber 1006 therebetween for receiving cells1008 and a liquid medium. The first substrate 1001 can be fabricatedfrom glass, Mylar, PDMS, silicon, a polymer, a semiconductor, or anycombination of them. The bioreactor 1000 further includes a secondsubstrate (not shown) sized such that when the second substrate 1050received by the first substrate 1001, the chamber 1006 is covered.

An inlet port (not shown) and a first connection channel 1021 are formedin the first substrate 1001, where the first connection channel 1021 isin fluid communication with the inlet port (not shown) and the chamber1006 for allowing a stream of substance to be delivered to the chamber1006.

Additionally, an outlet port (not shown) and a second connection channel1005 are formed in the first substrate 1001, where the second connectionchannel 1005 is in fluid communication with the outlet port (not shown)and the chamber 1006 for allowing a stream of substance to be removedfrom the chamber 1006.

The bioreactor 1000 further has confining means positioned in thechamber 1006 to form a confinement region 1006 a to confine the cells1008 therein. In one embodiment, the confining means includes a firstfilter 1085 a and a second filter 1085 b, where the first filter 1085 ais positioned proximate to the first connection channel 1021 and thesecond filter 1085 b is positioned proximate to the second connectionchannel 1005, and the first filter 1085 a and the second filter 1085 bare substantially parallel to each other. Each of the first filter 1085a and the second filter 1085 b includes a plurality of posts 1086 spacedapart from each other not to allow cells to pass through it. Thedistances between two neighboring posts can vary. For examples, posts1086, 1086 a, 1088 and 1089, as shown in FIGS. 10C, 10D, 10E and 10F,show posts with different gaps, respectively.

The first substrate 1001, referring now to FIG. 1C, further defines afirst alternate port 1083 and a third connection channel 1083 a that isin fluid communication with the first alternate port 1083 and theconfined region 1006 a of the chamber 1006 for allowing seed cells toperfuse only outside the confined region 1006 a in the chamber 1006.

The bioreactor 1100 further includes one or more supporting members 1082a, 1082 b positioned outside the confined region 1006 a of the chamber1006 for supporting the second substrate 1050. Additionally, thebioreactor 1100 further includes at least one supporting member 1087positioned inside the confined region 1006 a of the chamber 1006 forsupporting the second substrate 1050. Note that the chamber 1006 isformed with sidewalls of the chamber 1006 are tapered at theintersections of the connection channels with the chamber 1006 to forman angle of inclination α, which is preferred in the range of aboutbetween 10-45° from vertical, and an enclosed angle β, which ispreferred in the range of about between 30-80°, respectively, to avoidshear forces generated by sharp corners.

Bioreactor with Multiple Chambers

Referring now to FIGS. 8(A-D), the present invention can also bepracticed in association with an inventive bioreactor 800 and itsvariants as shown in FIGS. 8(A-D). In one embodiment, referring first toFIG. 8A, the bioreactor 800 includes a first substrate 801 having afirst surface and an opposite second surface, defining a first chamber812 therebetween for receiving a first type of cells and a liquidmedium. One or more second chambers 811 a, 811 b, 811 c, 811 d areformed in the first substrate 801 for receiving a second type of cellsand a liquid medium. Moreover, one or more connection channels 813 a,813 b, 813 c, 813 d are formed in the first substrate 801, wherein eachof connection channels 813 a, 813 b, 813 c, 813 d is in fluidcommunication with a corresponding second chamber 811 a, 811 b, 811 c,811 d and the first chamber 812 for allowing the first type of cells andthe second type of the cells to interact with each other. For example,connection channel 813 a is in fluid communication with a correspondingsecond chamber 811 a and the first chamber 812. The first type of cellsincludes protozoa, and the second type of cells includes bacteria.

The connection channels 813 a, 813 b, 813 c, 813 d are formed to allowprotozoa to travel therein. However, a variety of structures can beutilized to limit the mobility of protozoa for different applications.For examples, in an embodiment 804 as shown in FIG. 8B, a sizinglimiting or exclusion post 805 is utilized to limit the mobility ofprotozoa, which can be used to evaluate the mobility of protozoa.Alternatively, in an embodiment 807 as shown in FIG. 8C, one of theconnection channels 813 a, 813 b, 813 c, 813 d is formed with across-sectional dimension 808 variable along the length of theconnection channel is utilized to limit the mobility of protozoa, whichcan also be used to evaluate the mobility of protozoa. Moreover, in anembodiment 809 as shown in FIG. 8D, a barrier 810 positioned in aconnection channel is utilized for separation of bacteria and protozoa,which can be used to evaluate protozoa chemotaxis.

Bioreactors with Single Chamber and Substance Injection Capacity

Referring now to FIGS. 12(A-D), the present invention can be practicedin association with an inventive bioreactor 1200 and its variants asshown in FIGS. 12(A-D). In one embodiment, referring first to FIGS.12A1, 12A2 and 12A3, the bioreactor 1200 includes a first substrate 1202having a first surface 1202 a, an opposite second surface 1202 b andedges. The bioreactor 1200 further includes a second substrate 1201having a first surface 1201 a and an opposite second surface 1201 b,defining a cavity 1201 c with a bottom surface 1201 d, where the bottomsurface 1201 d is located therebetween the first surface 1201 a and thesecond surface 1201 b. The first surface 1202 a of the first substrate1202 is received by the second surface 1201 b of the second substrate1201 to cover the cavity 1201 c so as to form a chamber 1203 forreceiving cells and a liquid medium. The second substrate 1201 can befabricated from glass, Mylar, PDMS, silicon, a polymer, a semiconductor,or any combination of them.

A port 1209 is formed in the second substrate 1201 between the bottomsurface 1201 d and the first surface 1201 a of the second substrate 1201with a first opening 1209 a and an opposite, second opening 1209 b. Asformed, the port 1209 is in fluid communication with the chamber 1203through the first opening 1209 a to allow a stream of substance to beintroduced into the chamber 1203 through the port 1209 substantiallyalong a first direction 1210. The stream of substance is controlled soas to provide a gradient, or a concentration gradient of the substance,to the chamber 1203 at least around the first opening 1209 a. Indeed,the design of this inventive bioreactor allows a concentration gradientof the substance to be provided to and substantially felt by the entirechamber 1203. The stream of substance includes a substance affecting thegrowth of cells such as chemokine.

The second substrate 1201 further defines a third opening 1209 c and anopposite fourth opening 1209 d adapted for allowing a flow of liquid tobe introduced into the chamber 1203 through the third opening 1209 c andaway from the chamber 1203 through the fourth opening 1209 dsubstantially along a second direction 1208. As shown in FIGS. 12A1,12A2 and 12A3, the second direction 1208 is substantially perpendicularto the first direction 1210.

The bioreactor 1200 further includes a biocompatible coating layer 1205a applied to the bottom surface 1201 d of the second substrate 1201. Thebiocompatible coating layer 1205 a includes a material that may inhibitcell adhesion to the biocompatible coating layer, enhance cell adhesionto the biocompatible coating layer, or function as a fluorescent markeror indicator of the state of cells.

In forming the bioreactor 1200, the first surface 1202 a of the firstsubstrate 1202 and the second surface 1201 b of the second substrate1201 is spaced such that when a layer of cells 1206 grows on thebiocompatible coating layer 1205 a, a flow of liquid can flow in thechamber 1203 between the first surface 1202 a of the first substrate1202 and the layer of cells 1206 along the second direction 1208. Theflow of liquid is controlled so as to provide a known shear force to thelayer of cells 1206. The flow of liquid may be further controlled so asto provide perfusion and maintenance to the layer of cells 1206. Inother words, this flow can perfuse all cells in the chamber 1203, andcan be intermittent only as allowed by cell maintenance. Note that inmotion this flow crosses the concentration gradient of the substance inthe region proximate to the first opening 1209 a of the port 1209. Thecells can be any type of living cells, including, but not limited to,bacteria, protozoa, or both, normal cells, tumor cells, or anycombination of them. Cells can be introduced into a chamberindividually, in a collection of cells, or in the form of biofilm. Inone embodiment, a layer of endothelial cells grows on the chamber 1203.

Moreover, the first surface 1202 a of the first substrate 1202 and thesecond surface 1201 b of the second substrate 1201 are spaced to furtherallow at least one cell 1207 to migrate above the layer of cells 1206.The at least one cell to migrate can be a cell having a type same ordifferent from the type of the layer of cells 1206.

In an alternative embodiment of the present invention as shown in FIG.12B, the bioreactor 1200 further includes a layer of porous material1220 that is positioned on the bottom surface 1201 d of the secondsubstrate 1201. A biocompatible coating layer 1205 a can be applied tothe layer of porous material 1220 such that the layer of porous material1220 is between the biocompatible coating layer 1205 a and the bottomsurface 1201 d of the second substrate 1201. The biocompatible coatinglayer 1205 a includes a material that may inhibit cell adhesion to thebiocompatible coating layer, enhance cell adhesion to the biocompatiblecoating layer, or function as a fluorescent marker or indicator of thestate of cells.

In this embodiment, as shown in FIG. 12B, the first surface 1202 a ofthe first substrate 1202 and the second surface 1201 b of the secondsubstrate 1201 are spaced such that when a layer of cells 1206 grows onthe biocompatible coating layer 1205 a, a flow of liquid can flow in thechamber 1203 between the first surface 1202 a of the first substrate1202 and the layer of cells. The flow of liquid can also be controlledso as to provide a known shear force to the layer of cells. As suchformed, the chamber 1203 is divided by the biocompatible coating layer1205 a into two regions: an upper region for flow, and a lower regionfor cell extravasation and/or other cell activities.

The layer of porous material 1220 can include collagen, an extracellularmatrix, at least one cell culture scaffold supportive to the layer ofcells 1206, or any combination of them. The layer of porous material1220 may allow at least one cell 1221 to extravasate below the layer ofcells 1206.

The first substrate 1202 is at least partially optically transparent. Abiocompatible coating layer 1205 b may be applied to the first surface1202 a of the first substrate 1202, where the biocompatible coatinglayer 1205 b includes a material that may inhibit cell adhesion to thebiocompatible coating layer, enhance cell adhesion to the biocompatiblecoating layer, or function as a fluorescent marker or indicator of thestate of cells.

Referring now to FIGS. 12A1, 12A2, and 12A3, the first substrate 1202and the second substrate 1201 are substantially parallel to each otherand a plurality of posts 1204 are positioned between the first surface1202 a of the first substrate 1202 and the second surface 1201 b of thesecond substrate 1201 to substantially maintain a predeterminedseparation between the first surface 1202 a of the first substrate 1202and the second surface 1201 b of the second substrate 1201 to allowoptical detecting of dynamic activities of cells in the chamber 1203.The dynamic activities of cells in the chamber 1203 are detectablethrough optical detecting means such as high-resolution opticalmicroscope or a fluorescence-imaging device or both.

The predetermined separation between the first surface 1202 a of thefirst substrate 1202 and the second surface 1201 b of the secondsubstrate 1201 should be maintained with sufficient accuracy foraccurate optical measurements. To this end, the plurality of posts arepositioned in at least two rows, and wherein each row of posts has atleast two posts spaced from each other to form a stable supportstructure.

In alternative embodiments as shown in FIG. 12C, in addition to thedesigns and structures set forth above related to FIGS. 12A1, 12A2, 12A3and 12B, respectively, a bioreactor 1200 further includes perfusionmeans 1230 in fluid communication with the chamber 1203 to allowdiffusional exchange of nutrients and metabolic byproducts with thechamber 1203.

The perfusion means 1230 includes a nanofilter 1231 with a plurality ofpores 1232 in fluid communication with the chamber 1203, wherein thepores 1232 are sized to allow diffusional exchange of nutrients andmetabolic byproducts with the chamber 1203 and not to allow cells tomigrate across the nanofilter 1231. The pores 1232 may be further sizedto allow cells to perfuse through only by bi-directional diffusionthrough the nanofilter 1231 in a manner such that substantially no shearis generated by the perfusion of cells. In one embodiment, the pores1232 of the nanofilter 1231 are sized to have a dimension smaller than400 nanometers cross-sectionally.

The perfusion means 1230 further includes a perfusion supply network influid communication with the nanofilter 1231 through the pores 1232. Inone embodiment, the perfusion supply network includes a plurality ofperfusion channels 1233, each being in fluid communication with thenanofilter 1231 to allow bi-directional, diffusional exchange ofnutrients and metabolic byproducts with the nanofilter 1231 and beingdimensioned to minimize pressure drops along each perfusion channel 1233and to allow passive diffusional exchange of nutrients and metabolicbyproducts along each perfusion channel 1233.

The perfusion supply network further includes a plurality ofintermediate supply channels 1234, each being in fluid communicationwith a plurality of corresponding perfusion channel 1233 so as toprovide perfusate to the plurality of corresponding perfusion channel1233. Moreover, the perfusion supply network has a plurality ofintermediate return channels 1235, each being in fluid communicationwith a plurality of corresponding perfusion channel 1233 so as tocollect perfusate from the plurality of corresponding perfusion channel1233.

Additionally, the perfusion supply network further includes a pluralityof main supply channels 1236, each being in fluid communication with aplurality of corresponding intermediate supply channel 1234 so as toprovide perfusate to the plurality of corresponding intermediate supplychannel 1234, and a plurality of main return channels 1237, each beingin fluid communication with a plurality of corresponding intermediatereturn channel 1237 so as to collect perfusate from the plurality ofcorresponding intermediate return channel 1237.

Bioreactors with Multiple Chambers and Substance Injection Capacity

Referring now to FIGS. 13(A-F), the present invention can also bepracticed in association with an inventive bioreactor 1300 and itsvariants as shown in FIGS. 13(A-F). In one embodiment, referring firstto FIGS. 13A and 13B, the bioreactor 1300 includes a first substrate1302 having a first surface 1302 a, an opposite second surface 1302 band edges. The bioreactor 1300 further includes a second substrate 1301having a first surface 1301 a and an opposite second surface 1301 b,defining a cavity 1301 c with a bottom surface 1301 d, where the bottomsurface 1301 d is located therebetween the first surface 1301 a and thesecond surface 1301 b. The first surface 1302 a of the first substrate1302 is received by the second surface 1301 b of the second substrate1301 to cover the cavity 1301 c so as to form a chamber 1303 forreceiving cells and a liquid medium. The second substrate 1301 can befabricated from glass, Mylar, PDMS, silicon, a polymer, a semiconductor,or any combination of them.

The bioreactor 1300 further includes a filter 1351 dividing the chamber1303 into a first subchamber 1353 and a second subchamber 1354, whereinthe filter 1351 has a porosity to allow the first subchamber 1353 andthe second subchamber 1354 in fluid communication. Additionally, a port1309 is formed in the second substrate 1301 between the bottom surface1301 d and the first surface 1301 a of the second substrate 1301 with afirst opening 1309 a and an opposite, second opening 1309 b. As formed,the port 1309 is in fluid communication with the second subchamber 1354through the first opening 1309 a to allow a stream of substance to beintroduced into the chamber 1303 through the port 1309 substantiallyalong a first direction 1310. Similar to the embodiments shown in FIGS.12(A-D) and set forth above, the stream of substance is controlled so asto provide a gradient, or a concentration gradient of the substance, tothe chamber 1303 at least around the first opening 1309 a. Again, thedesign of this inventive bioreactor allows a concentration gradient ofthe substance to be provided to and substantially felt by the entirechamber 1303. The stream of substance includes a substance affecting thegrowth of cells such as chemokine.

The second substrate 1301 further defines a third opening 1309 c and anopposite fourth opening 1309 d adapted for allowing a flow of liquid tobe introduced into at least one of the first subchamber 1353 and thesecond subchamber 1354 through the third opening 1309 c and away from atleast one of the first subchamber 1353 and the second subchamber 1354through the fourth opening 1309 d substantially along a second direction1308. As shown in FIG. 13A, the second direction 1308 is substantiallyperpendicular to the first direction 1310. As discussed in more detailbelow, same or different flows of liquid can be introduced to one orboth of the first subchamber 1353 and the second subchamber 1354.

The filter 1351 has a first surface 1351 a that partially defines thefirst subchamber 1353 with the first surface 1302 a of the firstsubstrate 1302, and an opposite second surface 1351 b that partiallydefines the second subchamber 1354 with the second surface 1301 b of thesecond substrate 1301. The filter 1351 includes a perfusion membrane1351 c with a plurality of pores 1351 d to allow the filter 1351 to bein fluid communication with one or both of the first subchamber 1353 andthe second subchamber 1354. The pores 1351 d of the filter 1351 aresized to allow diffusional exchange of nutrients and metabolicbyproducts with one or both of the first subchamber 1353 and the secondsubchamber 1354 but not to allow cells to migrate across the filter1351. The pores 1351 d are further sized to allow cells to perfusethrough the filter 1351 only by bidirectional diffusion in a manner suchthat substantially no shear is generated by the perfusion of cells. Inone embodiment, the pores 1351 d of the filter 1351 are sized to have adimension smaller than 400 nanometers cross-sectionally. In a morepreferred embodiment, the pores 1351 d of the filter 1351 are sized tohave a dimension about 10 to 100 nanometers cross-sectionally.

The bioreactor 1300 further includes a plurality of posts 1352 a thatare strategically positioned between the first surface 1302 a of thefirst substrate 1302 and the first surface 1351 a of the filter 1351 tosubstantially maintain a predetermined separation between the firstsurface 1302 a of the first substrate 1302 and the first surface 1351 aof the filter 1351 to allow optical detecting of dynamic activities ofcells in the first subchamber 1353. Additionally, the bioreactor 1300includes a plurality of posts 1352 b that are strategically positionedbetween the second surface 1301 b of the second substrate 1301 and thesecond surface 1351 b of the filter 1351 to substantially maintain apredetermined separation between the second surface 1301 b of the secondsubstrate 1301 and the second surface 1351 b of the filter 1351 to allowoptical detecting of dynamic activities of cells in the secondsubchamber 1354.

The predetermined separation between the first surface 1302 a of thefirst substrate 1302 and the first surface 1351 a of the filter 1351 andthe predetermined separation between the second surface 1301 b of thesecond substrate 1301 and the second surface 1351 b of the filter 1351should be maintained with sufficient accuracy for accurate opticalmeasurements, respectively. To this end, the plurality of posts 1352 aand 1352 b are positioned in at least two rows, respectively, and whereeach row of posts has at least two posts spaced from each other to forma stable support structure. Posts 1352 a and 1352 b, as shown in FIG.13A, may be positioned away from each other.

As such formed, the bioreactor 1300 allows activities such as cellseeding and flow in the first subchamber 1353 (or the upper chamber) andthe second subchamber 1354 (or the lower chamber) to be controlledindependently, thereby allowing coculture on opposite sides of thefilter 1351 by inverting the device prior to adhesion of the cells inthe lower chamber. Alternatively, the upper cell population can be grownon the filter 1351 simultaneously as the other cell population grows onthe lower surface of the lower chamber. Thus, when a first flow ofliquid 1355 is introduced into the first subchamber 1353, the first flowof liquid 1355 can be controlled so as to provide a known shear force toa first layer of cells 1306 growing in the first subchamber 1353 on thefirst surface 1351 a side of the filter 1351 and an environment thatsimulates a vascular space in the first subchamber 1353. Jointly orindependently, a second flow of liquid 1356 can also be introduced intothe second subchamber 1354, and the second flow of liquid 1356 can becontrolled so as to provide an environment that simulates a tissue spacein the second subchamber 1354. The fact that first flow of liquid 1355and the second flow of liquid 1356 can be controlled independently fromeach other means, among other things, they can have different contents,different flow velocities, and/or different timing of flow.

Moreover, as such formed, the bioreactor 1300 allows growing and cultureof multiple layers (or populations) of cells therein. In addition to thefirst layer of cells 1306 growing in the first subchamber 1353, a secondlayer of cells 1307 is capable of growing in the second subchamber 1354on the second surface 1351 b side of the filter 1351. The first layer ofcells 1306 growing in the first subchamber 1353 and the second layer ofcells 1307 growing in the second subchamber 1354 can be same ordifferent.

In an alternative embodiment as shown in FIG. 13B, an extension portmember 1360 defining a channel therein is provided. As formed, theextension port member 1360 is positioned complimentarily to the port1309 such that the channel of the extension port member 1360 is in fluidcommunication with the port 1309 and the first subchamber 1353 to allowthe stream of substance to be directly introduced to the firstsubchamber 1353.

In yet another embodiment, referring now to FIGS. 13C and 13D, inaddition to the designs and structures set forth above particularlyrelated to FIGS. 13A and 13B, a bioreactor 1300 further includesperfusion means 1330 in fluid communication with at least one of thefirst subchamber 1353 and the second subchamber 1354 to allowdiffusional exchange of nutrients and metabolic byproducts with thechamber 1303.

Similar to the perfusion means 1230 discussed above related toembodiments as shown in FIGS. 12C and 12D, respectively, the perfusionmeans 1330 includes a second filter (or nanofilter) 1331 (the filter1351 described above is considered as a first filter) with a pluralityof pores 1332 in fluid communication with the second subchamber 1354,wherein the pores 1332 are sized to allow diffusional exchange ofnutrients and metabolic byproducts with the second subchamber 1354 andnot to allow cells to migrate across the second filter 1331. The pores1332 of the second filter 1331, for example, can be sized to have adimension smaller than 400 nanometers cross-sectionally. The firstfilter 1351 and the second filter 1331 can be same or different.

The perfusion means 1330 further includes a perfusion supply network influid communication with the second filter 1331 through the pores 1332.In one embodiment, the perfusion supply network includes a plurality ofperfusion channels 1333, each being in fluid communication with thesecond filter 1331 to allow bi-directional, diffusional exchange ofnutrients and metabolic byproducts with the second filter 1331 and beingdimensioned to minimize pressure drops along each perfusion channel 1333and to allow passive diffusional exchange of nutrients and metabolicbyproducts along each perfusion channel 1333.

The perfusion supply network additionally includes a plurality ofintermediate supply channels 1334, each being in fluid communicationwith a plurality of corresponding perfusion channel 1333 so as toprovide perfusate to the plurality of corresponding perfusion channel1333. Moreover, perfusion supply network includes a plurality ofintermediate return channels 1335, each being in fluid communicationwith a plurality of corresponding perfusion channel 1333 so as tocollect perfusate from the plurality of corresponding perfusion channel1333.

Furthermore, the perfusion supply network includes a plurality of mainsupply channels 1336, each being in fluid communication with a pluralityof corresponding intermediate supply channel 1334 so as to provideperfusate to the plurality of corresponding intermediate supply channel1334, and a plurality of main return channels 1337, each being in fluidcommunication with a plurality of corresponding intermediate returnchannel 1337 so as to collect perfusate from the plurality ofcorresponding intermediate return channel 1337.

This type of bioreactor with a microfabricated transwell chamber withchemokine injection capability as shown in FIGS. 13C and 13D,respectively, can be utilized to supported a coculture with filterperfusion to allow independent control of perfusion and shear in thechamber. The perfusion means 1330 maintains the viability of cells inthe lower chamber 1354 independent of the flow in the upper chamber1353. Stream of substances affecting growth of cells such as chemotacticagents can be injected through dedicated ports in the perfusion means1330. Perfusion gradients can be created, for example, with appropriateparts of the perfusion supply network. This type of bioreactor allows,among other things, examination of intravasation, extravasation, andcell migration in solid tissue. For examples, as shown in FIG. 13D, amatrix 1372 of host cells may be cultured in collagen, matrigel, orother substrates in a tissue space 1320 (the lower chamber of thechamber 1303), a neutrophil or other cell 1373 may be extravasating froma vascular space (the upper chamber 1353 of the chamber 1303) into thetissue space 1320, one or more tumor cells 1374 grow in the tissue space1320, and a macrophage or other cell 1375 in the tissue space1320.Optionally, as shown in FIG. 13E, at least one insertion member1381 defining a cavity 1382 therein is provided. The insertion member1381 has a length L and is positioned through the second substrate 1301and into the tissue space 1320 such that the cavity 1382 of theinsertion member 1381 is in fluid communication with the firstsubchamber 1353 or the vascular space.

Correspondingly, a plug 1384 having a first surface 1384 a and anopposite second surface 1384 b is provided. The plug 1384 iscomplimentary to a corresponding insertion member 1381 such that whenthe plug 1384 is received into the cavity 1382 of the correspondinginsertion member 1381, the plug 1384 engages with the body of thecorresponding insertion member 1381 to seal the cavity 1382 and a volume1382 a is formed between the first surface 1384 a and the first filter1351 to allow a collection of cells to be received therein. Forexamples, a collection of tumor cells 1387 can be contained in thevolume 1382 a. Optionally, a cage adapted for separating the tumor cells1387 from the first subchamber 1353 can be utilized.

Additionally, the plug 1384 further defines a port 1385 in fluidcommunication with the volume 1382 a for injecting or withdrawing astream of substance affecting the growth of the tumor cells 1387 such aschemokine. Moreover, a plurality of electrodes 1389 adapted forelectrochemical measurements of the tumor cells 1387 can be utilizedtogether with the plug 1384 to form a metabolic sensing head.

In yet another alternative embodiment shown in FIG. 13F, a bioreactor1300 is provided with an extension port member 1392 defining a channeltherein. The extension port member 1392 is positioned such that thechannel of the extension port member 1392 is in fluid communication withthe first subchamber 1353 to allow a stream of substance is introducedto the first subchamber 1353 or the vascular space. For example, agradient 1393 of chemokine can be introduced into the first subchamber1353 or the vascular space. A similar structure 1395 can be utilized toprovide a stream of substance such as a gradient 1396 of chemokine tothe tissue space 1320.

This type of bioreactor allows, among other things, examination ofintravasation, extravasation, and cell migration in solid tissue and isequipped for tumor cell or explant seeding and the delivery of chemokinein both the tissue and vascular space. The ability to add tumor cells ortumor blocks, removed from a target such as a mouse, to the host tissuematrix will provide metastasis assay that could be used in highthroughput screening of anticancer drugs. Metabolic electrodes allowmeasurement of the local environment within the tumor. The tumorinjection port 1391 provides control of the local tumor environment andthe infusion of chemokine or drugs. Many other dynamics of cells can beobserved. For examples, as shown in FIG. 13F, a tumor cell 1394 is seento migrate out of the tissue space 1320 to the vascular space 1353,while another tumor cell 1397 maybe extravasating from the vascularspace 1353 into the tissue space 1320.

Nanofilter Fabrication and Perfusion Network

In the embodiment of various bioreactors of the present invention setforth above, at least some of them are shown with nanofilter, ornanopore filter, perfusion means or system and sensing elements forelectrochemical detection. These components can be fabricated entirelyusing standard microfabrication techniques, including patterning,etching, thin film deposition, and soft-lithography techniques. Variousfabrication methods in the art can be utilized. In one embodiment of theinvention, soft lithography techniques are utilized to fabricate thedevices using poly-dimethylsiloxane (PDMS) and replicationmolding^(66,67). The finished PDMS chip is fused to different PDMSmodules for 3-dimensional bioreactors or rigid substrates (e.g.,microscope cover slip, microelectrode bearing glass slide)⁶⁸. PDMS is abiocompatible material with an oxygen permeability comparable towater⁶⁹.

One advantage of the perfusion through a nanopore filter is that thecells are perfused only by bidirectional diffusion through the membrane,not by mass-transport, so there is no shear. As shown in FIGS. 9(A-D),in one embodiment, the present invention provides a multi-layer fractalperfusion system 900 that ensures that chemokines released by cells inthe matrix are not carried to other cells by the perfusion system—theperfusate which has been enriched by cell products is immediatelycarried away, where it cannot affect other cells and could also beanalyzed without further dilution. Additionally, the perfusion networkcan be designed to provide a concentration gradient of a desiredchemokine across the bioreactor.

Makine of Perfusion Membrane. Polycarbonate filters with pore sizesbelow 400 nm can successfully block the passage of cells, as would berequired for a cellular perfusion system wherein transfilter cellularmigration was not desired. Typical filters with 10 to 400 nm diameterpores are six microns thick and have a pore density of approximately6×10¹² to 1×10¹² pores/m²; which corresponds to pore areas of 0.2% to12% of the filter surface area, respectively. The low porosity of thefilters with the smallest holes will limit their applicability for highefficiency diffusional transport that is driven only by a concentrationgradient (in contrast to convective transport that can be driven by ahigh pressure differential across the filter). To create a perfusionsystem that relies only upon diffusion and hence eliminates shearforces, the present invention uses highly permeable nanopore filterswith typical pore sizes of 10-100 nm that have not previously been usedfor cell culture. Patterns of this size cannot be generated byphotolithography, but can be generated by self-assembly of blockcopolymer films⁷⁰. The PS-b-PMMA copolymer has been shown suitable forself-generation of the pattern, and transfer of the pattern to siliconnitride, silicon oxide and silicon^(61,71,72). This polymer processproduces polycrystalline, hexagonal close-packed arrays of pores with amean pore diameter of 20 nm and center-to-center spacing of 42 nm. Thestandard deviation on each of these is about 10% in an optimizedtemplate. Typically, the uniformity does not degrade significantlythrough the pattern transfer and the reactive ion etching processrequired to convert a pattern in polymer to a filter in silicon nitrideor alumina. Based upon the published data, the inventors make theconservative estimate of a final standard deviation of about 15%. Theresulting porosity of these films is quite remarkable. The pore densitycan be estimated as follows: if e is the center-to-center distancebetween pores and we assume that for hexagonal close packing of threepores per unit cell, then the unit cell area is 3 l²sin (60°), and thepore density is 1/0.87 l² or 1.15/l². Using the 20 nm diameter and 42 nmseparation, one has a pore density of 6.5×10¹⁴/m² if the array hasperfect, single-crystal hexagonal packing. Given the two-to-onerelationship between pore separation and pore diameter, it follows that67% of the membrane surface is covered by pores, a factor of 3,000higher than can be achieved with standard polycarbonate filters with 20nm pores. Defects may change these two numbers of course, but not bymuch. Hence 80 nm and 400 nm pore spacings can achieve pore densities of1.8×10¹⁴ and 7.2×10¹² pores/m², respectively, but will have the samefractional pore area.

FIG. 9F shows an atomic force micrograph of apolystyrene-b-polymethylmethacrylate (PS-b-PMMA) pattern (with the PMMAremoved) that the inventors have created on a silicon template. Patternssuch as this are used to create the hole mask for the perfusion membraneto be fabricated on a silicon-nitride-coated, double-sided, polishedsilicon wafer by means of reactive ion etching. Photolithography is thenbe used to define a thin film Pt pattern for the capillary network onthe opposite side of the silicon wafer. In a subsequent chemical etchingstep the silicon is removed where it is not protected by the metal film.Since the silicon nitride is inert it acts as an etch stop. Thistechnique is generally used in MEMS fabrication and is well established.The net result is be a series of 10 μm wide by one millimeter longchannels that are spanned by a sub-micron-thick silicon nitride filmthat has more that 60% of its area covered by uniform, 20 nm diameterpores. The 3,000-fold greater pore area and a membrane thickness that is10⁻¹-10⁻² that of the polycarbonate filter will provide a filter that isremarkably permeable to passive diffusional exchange as compared toconventional filters, yet has pore sizes that are sufficiently small toblock cells from migrating across the filter. Two more scanning electronmicrographs at two magnifications, image 970 for UV acet 3 06.TIF andimage 972 for UV acet 3 11.TIF, of a PS-b-PMMA film depositied onsilicon are shown in FIGS. 9E1 and 9E2, respectively.

Silicon nitride was chosen for the first membrane material because thefabrication of these structures has been demonstrated and the MEMSfabrication steps are well known. However, there may be advantages tousing the polymer templates for membranes. For example, polymermembranes could be functionalized to enhance the growth of endothelialcells. A variety of structures are possible with self-assembly innumerous polymer systems: either by spinodal decomposition⁷³ orcrystallization⁷⁴. Other polymer systems will be surveyed and modeledfor use as perfusion membranes and the most likely candidates selectedfor further study.

Making of Capillary Perfusion Network. The next step in building such abioreactor is to connect one end of each of the short channels toperfusate supply and the other end to a drain. In one embodiment of thepresent invention, a novel, multi-layer capillary perfusion network isutilized, where every layer of which contains an “arterial” branch forthe delivery of culture media uniformly across the back of a filtermembrane, and a “venous” branch to collect the solution that hasundergone diffusional exchange with the cells on the other side. A 3-Dnetwork can be fabricated from stacked mylar layers. Fluid is injectedat a single inlet, this inlet stream is split multiple times into anarray of capillaries that cover the membrane, and the fluid is thengathered by the network back into a single outlet stream. Fluid enteringthe network has a multitude of path choices, and the key to providinguniform flow through the capillaries is to ensure that the flowresistance is identical through all of these possible paths. One designincludes 4 layers of channels and one layer containing the inlet andoutlet ports. This network takes fluid from the 0.5 mm square inlet andsplits it into 51 capillaries, each of which is 50 microns square by1100 microns long. The capillary array covers a membrane area that is2.25 mm square, and the four channel layers, each formed by lasercutting of mylar sheets, have a total thickness of 750 microns. A largerarea can be covered by tiling the membrane with this pattern and addingadditional layers to join the pieces together. One feature of thenetwork design is to split channels symmetrically and keep channelsidentical within a layer. The crossing of short channels simplifiesalignment and mechanical support of the perfusion network, in that eachlayer maintains its structural rigidity. This approach works on allsupporting channels but those on the outermost channels in thesmallest-channel layers, which may have to be made narrower than theothers on a layer in order to maintain (nearly) identical flow in eachof the capillaries. To have the same flow in each capillary identical,the size of the capillaries must vary.

The elimination of shear forces can be accomplished by using only ahigh-permeability filter to separate the cells from a perfusion bath.The addition of the multilayer perfusion network of this inventionoffers a number of distinct advantages over this approach: It ispossible to choose between uniform perfusion gradient perfusion by thechoice of the perfusion network design or nutrient supply configuration,there is automatic separation of the supply from reaction products; thenetwork provides distributed mechanical support of ultrathin membrane;chemokines can be introduced at edge or at an interior point in thearray; it is possible to simulate regional annoxia by blocking channelsat the arteriole level (either transiently or permanently), the separateperfusion and flow channels allow independent control of the celldelivery and cell perfusion systems; the capillary network could, initself, be coated with endothelial cells; the network allows greatercontrol of the perfusion environment as required for co-cultures;time-resolved exudate sampling is limited only by the time required foranalytes to diffuse over short distances; there are no pressuregradients from bath convection or stirring; this approach provides arealistic model of tissue perfusion; it is possible to deliverdrugs/toxins/nutrients capable of complex time modulation in a rapid andspatially-uniform manner; there is no mixing on input and outputstreams, and the perfusion network does not act as a macroscopicelectrical short, but instead provides an anisotropic, short-scalecoupling that can benefit either electroporation for delivery of geneticmaterial, or the study of syncytial tissue such as the heart.

In one embodiment of the present invention, referring now to FIGS.9(A-C), 9D1 and 9D2, respectively, a layered perfusion system 900 isprovided for use in a bioreactor such as bioreactor 1300 as shown inFIG. 13C, where the bioreactor defines a chamber for receiving cells andliquid medium. The layered perfusion system 900 includes a filter 903,which is corresponding to the filter 1331 as shown in FIG. 13C, having afirst surface 903 a and an opposite, second surface 903 b and aplurality of pores 903 c defined therein. The layered perfusion system900 further has a first perfusion system layer 904 having a firstsurface 904 a and an opposite, second surface 904 b and a plurality ofperfusion channels 904 c defined therein, where the first surface 904 aof the first perfusion system layer 904 is received by the secondsurface 903 b of the filter 903 such that each of the plurality ofperfusion channels 904 c is in fluid communication with the filter 903to allow bi-directional, diffusional exchange of nutrients and metabolicbyproducts with the filter 903 as shown in FIG. 9D 1, where the flow 912in a perfusion channel 904 c is substantially in a direction A and thediffussion 913 cross the filter 903 through the pores 903 c isbi-directional along a direction B, which is perpendicular to A. Inother words, the difussion can take place in a direction 913 a out andin an opposite direction 913 b in. Furthermore, each perfusion channel904 c is dimensioned to minimize pressure drops along each perfusionchannel 904 c and to allow passive diffusional exchange of nutrients andmetabolic byproducts along each perfusion channel 904 c.

The layered perfusion system 900 further has a second perfusion systemlayer 905 having a first surface 905 a and an opposite, second surface905 b and a plurality of perfusion supply and return channels 905 cdefined therein, wherein the first surface 905 a of the second perfusionsystem layer 904 is received by the second surface 904 b of the firstperfusion system layer 904 such that each of the plurality of perfusionsupply and return channels 905 c is in fluid communication with at leastone of the plurality of perfusion channels 904 c, and wherein theplurality of perfusion supply and return channels 904 c are formed alonga direction substantially perpendicular to that of the plurality ofperfusion channels 904 c.

The layered perfusion system 900 also has a third perfusion system layer906 having a first surface 906 a and an opposite, second surface 906 band a plurality of intermediate supply and return channels 906 c definedtherein, wherein the first surface 906 a of the third perfusion systemlayer 906 is received by the second surface 905 b of the secondperfusion system layer 905 such that each of the plurality ofintermediate supply and return channels 906 c is in fluid communicationwith at least one of the plurality of perfusion supply and returnchannels 905 c, and wherein the plurality of intermediate supply andreturn channels 906 c are formed along a direction substantiallyperpendicular to that of the plurality of perfusion supply and returnchannels 905 c.

The layered perfusion system 900 further has a fourth perfusion systemlayer 907 having a first surface 907 a and an opposite, second surface907 b and a plurality of main supply and return channels 907 c definedtherein, wherein the first surface 907 a of the fourth perfusion systemlayer 907 is received by the second surface 906 b of the third perfusionsystem layer 906 such that each of the plurality of main supply andreturn channels 907 c is in fluid communication with at least one of theplurality of intermediate supply and return channels 906 c, and whereinthe plurality of main supply and return channels 907 c are formed alonga direction substantially perpendicular to that of the plurality ofintermediate supply and return channels 906 c.

The layered perfusion system 900 additionally has a fifth perfusionsystem layer 911 having a first surface 911 a and an opposite, secondsurface 911 b and a supply channel 911 c and a return channel 911 ddefined therein, wherein the first surface 911 a of the fifth perfusionsystem layer 911 is received by the second surface 907 b of the fourthperfusion system layer 907 such at both of supply and return channels911 c, 911 d are in fluid communication with at least one of theplurality of main supply and return channels 907 c, respectively andwherein the supply and return channels 911 c, 911 d are formed along adirection substantially perpendicular to that of the plurality of mainsupply and return channels 907 c.

The layered perfusion system 900 further has a supply port 908 defininga channel 908 a in fluid communication with the supply channel 911 c,and a drain port 909 defining a channel 909 a in fluid communicationwith the return channel 911 d.

In use, the filter 903 is in fluid communication with the chamber of abioreactor and each of the plurality of perfusion channels 904 c is influid communication with the filter 903 to allow bi-directional,diffusional exchange of nutrients and metabolic byproducts with thechamber of the bioreactor through the pores 903 c of the filter 903.

The pores 903 c are sized to allow diffusional exchange of nutrients andmetabolic byproducts with the chamber and not to allow cells to migrateacross the filter 903, wherein the pores 903 c of the second filter 903are sized to have a dimension smaller than 400 nanometerscross-sectionally.

As such formed, the layered perfusion system 900 can be used asperfusion network 1330 in association with bioractor 1300 as shown inFIG. 13C.

Referring now to FIG. 9D 3 and FIGS. 9G(1-5), the present inventionprovides a method for preparing a layered perfusion system for use in abioreactor. In one embodiment, the method includes arranging a siliconwafer 953, a silicon-nitride layer 952, and a coblock polymer layer 951such that the silicon-nitride layer 952 is positioned between thesilicon wafer 953 and the coblock polymer layer 951 to form a material950, as shown in FIG. 9G 1. Then a plurality of channels 904 c areetched in the silicon wafer 953, as shown in FIG. 9G 2. Then, thecoblock polymer layer 951 is patterned to form a plurality of openingcorresponding to positions where the plurality of pores 903 c are to beformed, as shown in FIG. 9G 2. Last, a plurality of pores 903 c areetched through the silicon-nitride layer 952 to form a filter 903 suchthat the plurality of pores are in fluid communication with theplurality of channels 904 c. The structure formed by this process isshown in FIG. 9G 5.

While there has been shown various embodiments of the present invention,it is to be understood that certain changes can be made in the form andarrangement of the elements of the apparatus and steps of the methods topractice the present invention as would be known to one skilled in theart without departing from the underlying scope of the invention as isparticularly set forth in the Claims. Furthermore, the embodimentsdescribed above are only intended to illustrate the principles of thepresent invention and are not intended to limit the claims to thedisclosed elements. Indeed, since many embodiments of the invention canbe made without departing from the spirit and scope of the invention,the invention resides in the claims hereinafter appended.

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1. A bioreactor comprising: a. a first substrate having a first surface,an opposite second surface and edges; b. a second substrate having afirst surface and an opposite second surface, defining a cavity with abottom surface, wherein the bottom surface is located therebetween thefirst surface and the second surface, and wherein the first surface ofthe first substrate is received by the second surface of the secondsubstrate to cover the cavity so as to form a chamber for receivingcells and a liquid medium; and c. a port formed between the bottomsurface and the first surface of the second substrate with a firstopening and an opposite, second opening, wherein the port is in fluidcommunication with the chamber through the first opening to allow astream of substance to be introduced into the chamber through the portsubstantially along a first direction.
 2. The bioreactor of claim 1,wherein the second substrate further defines a third opening and anopposite fourth opening adapted for allowing a flow of liquid to beintroduced into the chamber through the third opening and away from thechamber through the fourth opening substantially along a seconddirection, and wherein the second direction is substantiallyperpendicular to the first direction.
 3. The bioreactor of claim 2,further comprising a biocompatible coating layer applied to the bottomsurface of the second substrate.
 4. The bioreactor of claim 3, whereinthe biocompatible coating layer comprises a material that may inhibitcell adhesion to the biocompatible coating layer, enhance cell adhesionto the biocompatible coating layer, or function as a fluorescent markeror indicator of the state of cells.
 5. The bioreactor of claim 3,wherein the first surface of the first substrate and the second surfaceof the second substrate is spaced such that when a layer of cells growson the biocompatible coating layer, the flow of liquid can flow in thechamber between the first surface of the first substrate and the layerof cells.
 6. The bioreactor of claim 5, wherein the flow of liquid iscontrolled so as to provide a known shear force to the layer of cells.7. The bioreactor of claim 5, wherein the flow of liquid is controlledso as to provide perfusion and maintenance to the layer of cells.
 8. Thebioreactor of claim 5, wherein the cells comprise bacteria.
 9. Thebioreactor of claim 5, wherein the cells comprise protozoa.
 10. Thebioreactor of claim 5, wherein the cells comprise endothelial cells. 11.The bioreactor of claim 5, wherein the first surface of the firstsubstrate and the second surface of the second substrate is spaced tofurther allow at least one cell to migrate above the layer of cells. 12.The bioreactor of claim 11, wherein the at least one cell to migrate isa cell different from the layer of cells.
 13. The bioreactor of claim12, wherein the at least one cell to migrate is a cell same as the layerof cells.
 14. The bioreactor of claim 2, further comprising a layer ofporous material positioned on the bottom surface of the secondsubstrate.
 15. The bioreactor of claim 14, further comprising abiocompatible coating layer applied to the layer of porous material suchthat the layer of porous material is between the biocompatible coatinglayer and the bottom surface of the second substrate.
 16. The bioreactorof claim 3, wherein the biocompatible coating layer comprises a materialthat may inhibit cell adhesion to the biocompatible coating layer,enhance cell adhesion to the biocompatible coating layer, or function asa fluorescent marker or indicator of the state of cells.
 17. Thebioreactor of claim 3, wherein the first surface of the first substrateand the second surface of the second substrate are spaced such that whena layer of cells grows on the biocompatible coating layer, the flow ofliquid can flow in the chamber between the first surface of the firstsubstrate and the layer of cells.
 18. The bioreactor of claim 17,wherein the flow of liquid is controlled so as to provide a known shearforce to the layer of cells.
 19. The bioreactor of claim 18, wherein theflow of liquid is controlled so as to provide perfusion and maintenanceto the layer of cells.
 20. The bioreactor of claim 18, wherein the cellscomprise bacteria.
 21. The bioreactor of claim 18, wherein the cellscomprise protozoa.
 22. The bioreactor of claim 17, wherein the cellscomprise endothelial cells.
 23. The bioreactor of claim 17, wherein thefirst surface of the first substrate and the second surface of thesecond substrate are spaced to further allow at least one cell tomigrate above the layer of cells.
 24. The bioreactor of claim 23,wherein the at least one cell to migrate is a cell different from thelayer of cells.
 25. The bioreactor of claim 24, wherein the at least onecell to migrate is a cell same as the layer of cells.
 26. The bioreactorof claim 14, wherein the layer of porous material comprises collagen.27. The bioreactor of claim 14, wherein the layer of porous materialcomprises an extracellular matrix.
 28. The bioreactor of claim 14,wherein the layer of porous material comprises at least one cell culturescaffold supportive to the layer of cells.
 29. The bioreactor of claim14, wherein the layer of porous material allows at least one cell toextravasate below the layer of cells.
 30. The bioreactor of claim 1,wherein the second substrate is fabricated from glass, Mylar, PDMS,silicon, a polymer, a semiconductor, or any combination of them.
 31. Thebioreactor of claim 1, wherein the first substrate is at least partiallyoptically transparent.
 32. The bioreactor of claim 31, furthercomprising a plurality of posts positioned between the first surface ofthe first substrate and the second surface of the second substrate tosubstantially maintain a predetermined separation between the firstsurface of the first substrate and the second surface of the secondsubstrate to allow optical detecting of dynamic activities of cells inthe chamber.
 33. The bioreactor of claim 32, wherein the dynamicactivities of cells in the chamber are detectable through opticaldetecting means.
 34. The bioreactor of claim 33, wherein the opticaldetecting means comprises at least one of high-resolution opticalmicroscope and a fluorescence-imaging device.
 35. The bioreactor ofclaim 32, wherein the plurality of posts are positioned in at least tworows, and wherein each row of posts has at least two posts spaced fromeach other.
 36. The bioreactor of claim 32, wherein the first substrateand the second substrate are substantially parallel to each other. 37.The bioreactor of claim 31, further comprising a biocompatible coatinglayer applied to the first surface of the first substrate.
 38. Thebioreactor of claim 37, wherein the biocompatible coating layercomprises a material that may inhibit cell adhesion to the biocompatiblecoating layer, enhance cell adhesion to the biocompatible coating layer,or function as a fluorescent marker or indicator of the state of cells.39. The bioreactor of claim 1, wherein the stream of substance iscontrolled so as to provide a gradient to the chamber at least aroundthe first opening.
 40. The bioreactor of claim 39, wherein the stream ofsubstance comprises chemokine.
 41. The bioreactor of claim 40, whereinthe stream of substance comprises a substance affecting the growth ofcells.
 42. A bioreactor comprising: a. a first substrate having a firstsurface, an opposite second surface and edges; b. a second substratehaving a first surface and an opposite second surface, defining a cavitywith a bottom surface, wherein the bottom surface is locatedtherebetween the first surface and the second surface, and wherein thefirst surface of the first substrate is received by the second surfaceof the second substrate to cover the cavity so as to form a chamber forreceiving cells and a liquid medium; and c. perfusion means in fluidcommunication with the chamber to allow diffusional exchange ofnutrients and metabolic byproducts with the chamber.
 43. The bioreactorof claim 42, further comprising a port formed between the bottom surfaceand the first surface of the second substrate with a first opening andan opposite, second opening, wherein the port is in fluid communicationwith the chamber through the first opening to allow a stream ofsubstance to be introduced into the chamber through the portsubstantially along a first direction.
 44. The bioreactor of claim 43,wherein the second substrate further defines a third opening and anopposite fourth opening adapted for allowing a flow of liquid to beintroduced into the chamber through the third opening and away from thechamber through the fourth opening substantially along a seconddirection, and wherein the second direction is substantiallyperpendicular to the first direction.
 45. The bioreactor of claim 44,further comprising a biocompatible coating layer applied to the bottomsurface of the second substrate.
 46. The bioreactor of claim 45, whereinthe biocompatible coating layer comprises a material that may inhibitcell adhesion to the biocompatible coating layer, enhance cell adhesionto the biocompatible coating layer, or function as a fluorescent markeror indicator of the state of cells.
 47. The bioreactor of claim 45,wherein the first surface of the first substrate and the second surfaceof the second substrate is spaced such that when a layer of cells growson the biocompatible coating layer, the flow of liquid can flow in thechamber between the first surface of the first substrate and the layerof cells.
 48. The bioreactor of claim 47, wherein the flow of liquid iscontrolled so as to provide a known shear force to the layer of cells.49. The bioreactor of claim 47, wherein the flow of liquid is controlledso as to provide perfusion and maintenance to the layer of cells. 50.The bioreactor of claim 47, wherein the cells comprise bacteria.
 51. Thebioreactor of claim 47, wherein the cells comprise protozoa.
 52. Thebioreactor of claim 47, wherein the cells comprise endothelial cells.53. The bioreactor of claim 47, wherein the first surface of the firstsubstrate and the second surface of the second substrate is spaced tofurther allow at least one cell to migrate above the layer of cells. 54.The bioreactor of claim 53, wherein the at least one cell to migrate isa cell different from the layer of cells.
 55. The bioreactor of claim54, wherein the at least one cell to migrate is a cell same as the layerof cells.
 56. The bioreactor of claim 44, further comprising a layer ofporous material positioned on the bottom surface of the secondsubstrate.
 57. The bioreactor of claim 56, further comprising abiocompatible coating layer applied to the layer of porous material suchthat the layer of porous material is between the biocompatible coatinglayer and the bottom surface of the second substrate.
 58. The bioreactorof claim 57, wherein the biocompatible coating layer comprises amaterial that may inhibit cell adhesion to the biocompatible coatinglayer, enhance cell adhesion to the biocompatible coating layer, orfunction as a fluorescent marker or indicator of the state of cells. 59.The bioreactor of claim 57, wherein the first surface of the firstsubstrate and the second surface of the second substrate is spaced suchthat when a layer of cells grows on the biocompatible coating layer, theflow of liquid can flow in the chamber between the first surface of thefirst substrate and the layer of cells.
 60. The bioreactor of claim 59,wherein the flow of liquid is controlled so as to provide a known shearforce to the layer of cells.
 61. The bioreactor of claim 60, wherein theflow of liquid is controlled so as to provide perfusion and maintenanceto the layer of cells.
 62. The bioreactor of claim 61, wherein the cellscomprise bacteria.
 63. The bioreactor of claim 61, wherein the cellscomprise protozoa.
 64. The bioreactor of claim 61, wherein the cellscomprise endothelial cells.
 65. The bioreactor of claim 61, wherein thefirst surface of the first substrate and the second surface of thesecond substrate is spaced to further allow at least one cell to migrateabove the layer of cells.
 66. The bioreactor of claim 65, wherein the atleast one cell to migrate is a cell different from the layer of cells.67. The bioreactor of claim 66, wherein the at least one cell to migrateis a cell same as the layer of cells.
 68. The bioreactor of claim 56,wherein the layer of porous material comprises collagen.
 69. Thebioreactor of claim 56, wherein the layer of porous material comprisesan extracellular matrix.
 70. The bioreactor of claim 56, wherein thelayer of porous material comprises at least one cell culture scaffoldsupportive to the layer of cells.
 71. The bioreactor of claim 56,wherein the layer of porous material allows at least one cell toextravasate below the layer of cells.
 72. The bioreactor of claim 43,wherein the stream of substance is controlled so as to provide agradient to the chamber at least around the first opening.
 73. Thebioreactor of claim 72, wherein the stream of substance compriseschemokine.
 74. The bioreactor of claim 72, wherein the stream ofsubstance comprises a substance affecting the growth of cells.
 75. Thebioreactor of claim 42, wherein the second substrate is fabricated fromglass, Mylar, PDMS, silicon, a polymer, a semiconductor, or anycombination of them.
 76. The bioreactor of claim 42, wherein the firstsubstrate is at least partially optically transparent.
 77. Thebioreactor of claim 76, further comprising a plurality of postspositioned between the first surface of the first substrate and thesecond surface of the second substrate to substantially maintain apredetermined separation between the first surface of the firstsubstrate and the second surface of the second substrate to allowoptical detecting of dynamic activities of cells in the chamber.
 78. Thebioreactor of claim 77, wherein the dynamic activities of cells in thechamber are detectable through optical detecting means.
 79. Thebioreactor of claim 78, wherein the optical detecting means comprises atleast one of high-resolution optical microscope and afluorescence-imaging device.
 80. The bioreactor of claim 77, wherein theplurality of posts are positioned in at least two rows, and wherein eachrow of posts has at least two posts spaced from each other.
 81. Thebioreactor of claim 77, wherein the first substrate and the secondsubstrate are substantially parallel to each other.
 82. The bioreactorof claim 76, further comprising a biocompatible coating layer applied tothe first surface of the first substrate.
 83. The bioreactor of claim82, wherein the biocompatible coating layer comprises a material thatmay inhibit cell adhesion to the biocompatible coating layer, enhancecell adhesion to the biocompatible coating layer, or function as afluorescent marker or indicator of the state of cells.
 84. Thebioreactor of claim 42, wherein the perfusion means comprises: a. ananofilter with a plurality of pores in fluid communication with thechamber, wherein the pores are sized to allow diffusional exchange ofnutrients and metabolic byproducts with the chamber and not to allowcells to migrate across the nanofilter; and b. a perfusion supplynetwork in fluid communication with the nanofilter through the pores.85. The bioreactor of claim 84, wherein the pores are further sized toallow cells to perfuse through only by bidirectional diffusion throughthe nanofilter in a manner such that substantially no shear is generatedby the perfusion of cells.
 86. The bioreactor of claim 85, wherein theperfusion supply network comprises: a. a plurality of perfusionchannels, each being in fluid communication with the nanofilter to allowbidirectional, diffusional exchange of nutrients and metabolicbyproducts with the nanofilter and being dimensioned to minimizepressure drops along each perfusion channel and to allow passivediffusional exchange of nutrients and metabolic byproducts along eachperfusion channel; b. a plurality of intermediate supply channels, eachbeing in fluid communication with a plurality of corresponding perfusionchannels so as to provide perfusate to the plurality of correspondingperfusion channels; and c. a plurality of intermediate return channels,each being in fluid communication with a plurality of correspondingperfusion channels so as to collect perfusate from the plurality ofcorresponding perfusion channels.
 87. The bioreactor of claim 86,wherein the perfusion supply network further comprises: a. a pluralityof main supply channels, each being in fluid communication with aplurality of corresponding intermediate supply channels so as to provideperfusate to the plurality of corresponding intermediate supplychannels; and b. a plurality of main return channels, each being influid communication with a plurality of corresponding intermediatereturn channels so as to collect perfusate from the plurality ofcorresponding intermediate return channels.
 88. The bioreactor of claim84, wherein the pores of the nanofilter are sized to have a dimensionsmaller than 400 nanometers cross-sectionally.
 89. A bioreactorcomprising: a. a first substrate having a first surface, an oppositesecond surface and edges; b. a second substrate having a first surfaceand an opposite second surface, defining a cavity with a bottom surface,wherein the bottom surface is located therebetween the first surface andthe second surface, and wherein the first surface of the first substrateis received by the second surface of the second substrate to cover thecavity so as to form a chamber for receiving cells and a liquid medium;c. a filter dividing the chamber into a first subchamber and a secondsubchamber, wherein the filter has a porosity to allow the firstsubchamber and the second subchamber in fluid communication; and d. aport formed between the bottom surface and the first surface of thesecond substrate with a first opening and an opposite, second opening,wherein the port is in fluid communication with the second subchamberthrough the first opening to allow a stream of substance to beintroduced into the chamber through the port substantially along a firstdirection.
 90. The bioreactor of claim 89, wherein the second substratefurther defines a third opening and an opposite fourth opening adaptedfor allowing a flow of liquid to be introduced into at least one of thefirst subchamber and the second subchamber through the third opening andaway from at least one of the first subchamber and the second subchamberthrough the fourth opening substantially along a second direction, andwherein the second direction is substantially perpendicular to the firstdirection.
 91. The bioreactor of claim 90, wherein the filter has afirst surface that defines the first subchamber with the first surfaceof the first substrate, and an opposite second surface that defines thesecond subchamber with the second surface of the second substrate. 92.The bioreactor of claim 91, wherein the filter comprises a perfusionmembrane with a plurality of pores in fluid communication with at leastone of the first subchamber and the second subchamber, wherein the poresare sized to allow diffusional exchange of nutrients and metabolicbyproducts with at least one of the first subchamber and the secondsubchamber and not to allow cells to migrate across the filter.
 93. Thebioreactor of claim 92, wherein the pores are further sized to allowcells to perfuse through only by bi-directional diffusion through thefilter in a manner such that substantially no shear is generated by theperfusion of cells.
 94. The bioreactor of claim 93, wherein the pores ofthe filter are sized to have a dimension smaller than 400 nanometerscross-sectionally.
 95. The bioreactor of claim 91, further comprising aplurality of posts positioned between the first surface of the firstsubstrate and the first surface of the filter to substantially maintaina predetermined separation between the first surface of the firstsubstrate and the first surface of the filter to allow optical detectingof dynamic activities of cells in the first subchamber.
 96. Thebioreactor of claim 95, further comprising a plurality of postspositioned between the second surface of the second substrate and thesecond surface of the filter to substantially maintain a predeterminedseparation between the second surface of the second substrate and thesecond surface of the filter to allow optical detecting of dynamicactivities of cells in the second subchamber.
 97. The bioreactor ofclaim 96, wherein the plurality of posts are positioned in at least tworows, and wherein each row of posts has at least two posts spaced fromeach other.
 98. The bioreactor of claim 96, wherein when a first flow ofliquid is introduced into the first subchamber, the first flow of liquidis controlled so as to provide a known shear force to a first layer ofcells growing in the first subchamber on the first surface side of thefilter and an environment that simulates a vascular space in the firstsubchamber.
 99. The bioreactor of claim 98, wherein when a second flowof liquid is introduced into the second subchamber, the second flow ofliquid is controlled so as to provide an environment that simulates atissue space in the second subchamber.
 100. The bioreactor of claim 99,wherein the first flow of liquid and the second flow of liquid aredifferent.
 101. The bioreactor of claim 98, wherein a second layer ofcells is capable of growing in the second subchamber on the secondsurface side of the filter.
 102. The bioreactor of claim 101, whereinthe first layer of cells growing in the first subchamber and the secondlayer of cells growing in the second subchamber are different.
 103. Thebioreactor of claim 90, further comprising an extension port memberdefining a channel therein, wherein the extension port member ispositioned complimentary to the port such that the channel of theextension port member is in fluid communication with the port and thefirst subchamber to allow the stream of substance is introduced to thefirst subchamber.
 104. The bioreactor of claim 89, wherein the secondsubstrate is fabricated from glass, Mylar, PDMS, silicon, a polymer, asemiconductor, or any combination of them.
 105. The bioreactor of claim89, wherein the first substrate is at least partially opticallytransparent.
 106. The bioreactor of claim 89, wherein the stream ofsubstance is controlled so as to provide a gradient to the chamber atleast around the first opening.
 107. The bioreactor of claim 106,wherein the stream of substance comprises chemokine.
 108. The bioreactorof claim 106, wherein the stream of substance comprises a substanceaffecting the growth of cells.
 109. A bioreactor comprising: a. a firstsubstrate having a first surface, an opposite second surface and edges;b. a second substrate having a first surface and an opposite secondsurface, defining a cavity with a bottom surface, wherein the bottomsurface is located therebetween the first surface and the secondsurface, and wherein the first surface of the first substrate isreceived by the second surface of the second substrate to cover thecavity so as to form a chamber for receiving cells and a liquid medium;c. a first filter dividing the chamber into a first subchamber and asecond subchamber, wherein the first filter has a porosity to allow thefirst subchamber and the second subchamber in fluid communication; d.perfusion means in fluid communication with at least one of the firstsubchamber and the second subchamber to allow diffusional exchange ofnutrients and metabolic byproducts with the chamber; and e. a portformed between the bottom surface and the first surface of the secondsubstrate with a first opening and an opposite, second opening, whereinthe port is in fluid communication with the second subchamber throughthe first opening to allow a stream of substance to be introduced intothe chamber through the port substantially along a first direction. 110.The bioreactor of claim 109, further comprising a port formed betweenthe bottom surface and the first surface of the second substrate with afirst opening and an opposite, second opening, wherein the port is influid communication with the second subchamber through the first openingto allow a stream of substance to be introduced into the chamber throughthe port substantially along a first direction.
 111. The bioreactor ofclaim 110, wherein the second substrate further defines a third openingand an opposite fourth opening adapted for allowing a flow of liquid tobe introduced into at least one of the first subchamber and the secondsubchamber through the third opening and away from at least one of thefirst subchamber and the second subchamber through the fourth openingsubstantially along a second direction, and wherein the second directionis substantially perpendicular to the first direction.
 112. Thebioreactor of claim 109, wherein the first filter has a first surfacethat defines the first subchamber with the first surface of the firstsubstrate, and an opposite second surface that defines the secondsubchamber with the second surface of the second substrate.
 113. Thebioreactor of claim 112, wherein the first filter comprises a perfusionmembrane with a plurality of pores in fluid communication with at leastone of the first subchamber and the second subchamber, wherein the poresare sized to allow diffusional exchange of nutrients and metabolicbyproducts with at least one of the first subchamber and the secondsubchamber and not to allow cells to migrate across the first filter.114. The bioreactor of claim 113, wherein the pores are further sized toallow cells to perfuse through only by bidirectional diffusion throughthe first filter in a manner such that substantially no shear isgenerated by the perfusion of cells.
 115. The bioreactor of claim 114,wherein the pores of the first filter are sized to have a dimensionsmaller than 400 nanometers cross-sectionally.
 116. The bioreactor ofclaim 112, further comprising a plurality of posts positioned betweenthe first surface of the first substrate and the first surface of thefirst filter to substantially maintain a predetermined separationbetween the first surface of the first substrate and the first surfaceof the first filter to allow optical detecting of dynamic activities ofcells in the first subchamber.
 117. The bioreactor of claim 116, furthercomprising a plurality of posts positioned between the second surface ofthe second substrate and the second surface of the first filter tosubstantially maintain a predetermined separation between the secondsurface of the second substrate and the second surface of the firstfilter to allow optical detecting of dynamic activities of cells in thesecond subchamber.
 118. The bioreactor of claim 117, wherein theplurality of posts are positioned in at least two rows, and wherein eachrow of posts has at least two posts spaced from each other.
 119. Thebioreactor of claim 117, wherein when a first flow of liquid isintroduced into the first subchamber, the first flow of liquid iscontrolled so as to provide a known shear force to a first layer ofcells growing in the first subchamber on the first surface side of thefirst filter and an environment that simulates a vascular space in thefirst subchamber.
 120. The bioreactor of claim 119, wherein when asecond flow of liquid is introduced into the second subchamber, thesecond flow of liquid is controlled so as to provide an environment thatsimulates a tissue space in the second subchamber.
 121. The bioreactorof claim 120, wherein the first flow of liquid and the second flow ofliquid are different.
 122. The bioreactor of claim 119, wherein a secondlayer of cells is capable of growing in the second subchamber on thesecond surface side of the first filter.
 123. The bioreactor of claim122, wherein the first layer of cells growing in the first subchamberand the second layer of cells growing in the second subchamber aredifferent.
 124. The bioreactor of claim 109, wherein the perfusion meanscomprises: a. a second filter with a plurality of pores in fluidcommunication with the second subchamber, wherein the pores are sized toallow diffusional exchange of nutrients and metabolic byproducts withthe second subchamber and not to allow cells to migrate across thesecond filter; and b. a perfusion supply network in fluid communicationwith the second filter through the pores.
 125. The bioreactor of claim124, wherein the perfusion supply network comprises: a. a plurality ofperfusion channels, each being in fluid communication with the secondfilter to allow bi-directional, diffusional exchange of nutrients andmetabolic byproducts with the second filter and being dimensioned tominimize pressure drops along each perfusion channel and to allowpassive diffusional exchange of nutrients and metabolic byproducts alongeach perfusion channel; b. a plurality of intermediate supply channels,each being in fluid communication with a plurality of correspondingperfusion channels so as to provide perfusate to the plurality ofcorresponding perfusion channels; and c. a plurality of intermediatereturn channels, each being in fluid communication with a plurality ofcorresponding perfusion channels so as to collect perfilsate from theplurality of corresponding perfusion channels.
 126. The bioreactor ofclaim 125, wherein the perfusion supply network further comprises: a. aplurality of main supply channels, each being in fluid communicationwith a plurality of corresponding intermediate supply channels so as toprovide perfusate to the plurality of corresponding intermediate supplychannels; and b. a plurality of main return channels, each being influid communication with a plurality of corresponding intermediatereturn channels so as to collect perfusate from the plurality ofcorresponding intermediate return channels.
 127. The bioreactor of claim124, wherein the pores of the second filter are sized to have adimension smaller than 400 nanometers cross-sectionally.
 128. Thebioreactor of claim 124, wherein the first filter and the second filterare different.
 129. The bioreactor of claim 110, further comprising atleast one insertion member defining a cavity therein, wherein theinsertion member has a length L and is positioned through the secondsubstrate such that the cavity of the insertion member is in fluidcommunication with the first subchamber.
 130. The bioreactor of claim129, further comprising a plug having a first surface and an oppositesecond surface and complimentary to a corresponding insertion membersuch that when the plug is received into the cavity of the correspondinginsertion member, the plug engages with the body of the correspondinginsertion member to seal the cavity and a volume is formed between thefirst surface and the first filter to allow a collection of cells to bereceived therein.
 131. The bioreactor of claim 130, wherein thecollection of cells comprises tumor cells.
 132. The bioreactor of claim131, wherein the plug further defines a port in fluid communication withthe volume for injecting or withdrawing a stream of substance affectingthe growth of the tumor cells.
 133. The bioreactor of claim 129, furthercomprising a cage adapted for separating the tumor cells from the firstsubchamber.
 134. The bioreactor of claim 129, further comprising aplurality of electrodes adapted for electrochemical measurements of thetumor cells.
 135. The bioreactor of claim 109, further comprising anextension port member defining a channel therein, wherein the extensionport member is positioned such that the channel of the extension portmember is in fluid communication with the first subchamber to allow astream of substance is introduced to the first subchamber.
 136. Thebioreactor of claim 135, wherein the stream of substance is controlledso as to provide a gradient to the first subchamber.
 137. The bioreactorof claim 136, wherein the stream of substance comprises chemokine. 138.The bioreactor of claim 136, wherein the stream of substance comprises asubstance affecting the growth of cells.
 139. The bioreactor of claim109, wherein the second substrate is fabricated from glass, Mylar, PDMS,silicon, a polymer, a semiconductor, or any combination of them. 140.The bioreactor of claim 109, wherein the first substrate is at leastpartially optically transparent.
 141. The bioreactor of claim 109,wherein the stream of substance is controlled so as to provide agradient to the second subchamber.
 142. The bioreactor of claim 141,wherein the stream of substance comprises chemokine.
 143. The bioreactorof claim 141, wherein the stream of substance comprises a substanceaffecting the growth of cells.
 144. A layered perfusion system 900 foruse in a bioreactor, wherein the bioreactor defines a chamber forreceiving cells and liquid medium, comprises: a. a filter 903 having afirst surface 903 a and an opposite, second surface 903 b and aplurality of pores 903 c defined therein; and b. a perfusion supplynetwork in fluid communication with the filter 903 through the pores 903c.
 145. The bioreactor of claim 144, wherein the perfusion supplynetwork comprises a first perfusion system layer 904 having a firstsurface 904 a and an opposite, second surface 904 b and a plurality ofperfusion channels 904 c defined therein, wherein the first surface 904a of the first perfusion system layer 904 is received by the secondsurface 903 b of the filter 903 such that at each of the plurality ofperfusion channels 904 c is in fluid communication with the filter 903to allow bidirectional, diffusional exchange of nutrients and metabolicbyproducts with the filter 903 and is dimensioned to minimize pressuredrops along each perfusion channel 904 c and to allow passivediffusional exchange of nutrients and metabolic byproducts along eachperfusion channel 904 c.
 146. The bioreactor of claim 145, wherein theperfusion supply network further comprises a second perfusion systemlayer 905 having a first surface 905 a and an opposite, second surface905 b and a plurality of perfusion supply and return channels 905 cdefined therein, wherein the first surface 905 a of the second perfusionsystem layer 904 is received by the second surface 904 b of the firstperfusion system layer 904 such that each of the plurality of perfusionsupply and return channels 905 c is in fluid communication with at leastone of the plurality of perfusion channels 904 c, and wherein theplurality of perfusion supply and return channels 904 c are formed alonga direction substantially perpendicular to that of the plurality ofperfusion channels 904 c.
 147. The bioreactor of claim 146, wherein theperfusion supply network further comprises a third perfusion systemlayer 906 having a first surface 906 a and an opposite, second surface906 b and a plurality of intermediate supply and return channels 906 cdefined therein, wherein the first surface 906 a of the third perfusionsystem layer 906 is received by the second surface 905 b of the secondperfusion system layer 905 such that each of the plurality ofintermediate supply and return channels 906 c is in fluid communicationwith at least one of the plurality of perfusion supply and returnchannels 905 c, and wherein the plurality of intermediate supply andreturn channels 906 c are formed along a direction substantiallyperpendicular to that of the plurality of perfusion supply and returnchannels 905 c.
 148. The bioreactor of claim 147, wherein the perfusionsupply network further comprises a fourth perfusion system layer 907having a first surface 907 a and an opposite, second surface 907 b and aplurality of main supply and return channels 907 c defined therein,wherein the first surface 907 a of the fourth perfusion system layer 907is received by the second surface 906 b of the third perfusion systemlayer 906 such that each of the plurality of main supply and returnchannels 907 c is in fluid communication with at least one of theplurality of intermediate supply and return channels 906 c, and whereinthe plurality of main supply and return channels 907 c are formed alonga direction substantially perpendicular to that of the plurality ofintermediate supply and return channels 906 c.
 149. The bioreactor ofclaim 148, wherein the perfusion supply network further comprises afifth perfusion system layer 911 having a first surface 911 a and anopposite, second surface 911 b and a supply channel 911 c and a returnchannel 911 d defined therein, wherein the first surface 911 a of thefifth perfusion system layer 911 is received by the second surface 907 bof the fourth perfusion system layer 907 such at both of supply andreturn channels 911 c, 911 d are in fluid communication with at leastone of the plurality of main supply and return channels 907 c, andwherein the supply and return channels 911 c, 911 d are formed along adirection substantially perpendicular to that of the plurality of mainsupply and return channels 907 c.
 150. The bioreactor of claim 149,further comprising a supply port 908 defining a channel 908 a in fluidcommunication with the supply channel 911 c, and a drain port 909defining a channel 909 a in fluid communication with the return channel911 d.
 151. The bioreactor of claim 144, wherein the filter 903 is influid communication with the chamber of the bioreactor and each of theplurality of perfusion channels 904 c is in fluid communication with thefilter 903 to allow bi-directional, diffusional exchange of nutrientsand metabolic byproducts with the chamber of the bioreactor through thepores 903 c of the filter
 903. 152. The bioreactor of claim 144, whereinthe pores 903 c are sized to allow diffusional exchange of nutrients andmetabolic byproducts with the chamber and not to allow cells to migrateacross the filter
 903. 153. The bioreactor of claim 144, wherein thepores 903 c of the second filter 903 are sized to have a dimensionsmaller than 400 nanometers cross-sectionally.
 154. A method forpreparing a layered perfusion system 900 for use in a bioreactor,wherein the bioreactor defines a chamber for receiving cells and liquidmedium, comprises the steps of: a. arranging a silicon wafer 953, asilicon-nitride layer 952, and a coblock polymer layer 951 such that thesilicon-nitride layer 952 is positioned between the silicon wafer 953and the coblock polymer layer 951; b. etching a plurality of channels904 c in the silicon wafer 953; and c. etching a plurality of pores 903c through the silicon-nitride layer 952 to form a filter 903 such thatthe plurality of pores are in fluid communication with the plurality ofchannels 904 c.
 155. The bioreactor of claim 154, prior to the step ofetching a plurality of pores 903 c, further comprising a step ofpatterning the coblock polymer layer 951 to form a plurality of openingcorresponding to positions where the plurality of pores 903 c are to beformed.